Biomarkers for graft-versus-host disease

ABSTRACT

Biomarkers are provided that are predictive of a subject&#39;s responsiveness to a therapy comprising a JAK inhibitor. The biomarkers, compositions, and methods described herein are useful in selecting appropriate treatment modalities for a subject having, suspected of having, or at risk of developing Graft-Versus-Host Disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Appl. No. 62/657,193, filed Apr. 13, 2018, and U.S. Provisional Appl. No. 62/773,308, filed Nov. 30, 2018. The content of the prior applications are incorporated by reference herein in their entirety.

TECHNICAL FIELD

The present invention relates generally to biomarkers and Graft-Versus-Host Disease.

BACKGROUND

Graft-Versus-Host Disease (GvHD) occurs when immunologically competent cells transferred to an allogeneic recipient attack tissues in the recipient. Tissues of the skin, gut epithelia, and liver are often targets and may be destroyed during the course of GvHD. The disease presents an especially severe problem when immune tissue is being transplanted, such as in bone marrow transplantation. GvHD is the second leading cause of death following allogeneic hematopoietic stem cell transplant. GvHD can also occur following other transplants, such as heart and liver transplants.

Janus kinase (JAK) inhibitors have been developed as agents for the treatment of GvHD. However, as for any therapeutic, JAK inhibitors may not be equally effective in all subjects that have GvHD. There is a need for means of identifying those subjects having GvHD that could most benefit from treatment with a JAK inhibitor as well as identifying those subjects that exhibit a therapeutic response to treatment with a JAK inhibitor.

SUMMARY

The present application is based, at least in part, on the identification of biomarkers that are predictive of a GvHD subject's responsiveness to a therapy comprising a JAK inhibitor and biomarkers that identify a subject that has undergone a therapeutic response to a JAK inhibitor. The level of certain proteins (e.g., the proteins listed in Table 1 and Table 2) prior to treatment is identified as a useful predictor of responsiveness to a therapy comprising a JAK inhibitor. In addition, the change in level of certain proteins (e.g., the proteins listed in Table 13) during the course of treatment is identified as a useful identifier of responsiveness to a therapy comprising a JAK inhibitor. Thus, the biomarkers and compositions described herein are useful, for example, in identifying, stratifying, and/or selecting a patient or a subset of patients having, suspected of having, or at risk of developing GvHD that could benefit, or have benefitted, from treatment with a JAK inhibitor. In addition, the methods described herein are useful, for example, in selecting appropriate treatment modalities (e.g., therapy comprising a JAK inhibitor) for a subject suffering from, suspected of having, or at risk of developing GvHD.

The disclosure features a method of treating a human subject having, suspected of having, or at risk of developing GvHD by administering to the human subject a therapy comprising a JAK inhibitor, wherein the human subject has been previously determined to have (i) a baseline concentration of at least one protein (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 proteins) selected from the group consisting of IL8, HAOX1, ENPP7, ACE2, SULT2A1, MCP-3, CES1, MFGE8, PLXNB1, TNFRSF10A, CCL15, SEMA4C, PREB, NFATC3, CCL19, DLL1, ENTPD2, IL-4RA, EPHA2, FOSB, CXCL10, VAMP5, ALDH3A1, MVK, IL12RB1, CALCA, AHCY, PRSS2, LILRB4, DDAH1, IL-1ra, NECTIN2, PDCD1, CD74, PD-L1, REG3A, CA5A, N2DL-2, CDCP1, U-PAR, SIGLEC7, ANGPTL4, ALDH1A1, SPINK1, HTRA2, PRDX6, IL-1RT2, IGFBP-1, HNMT, TRAIL-R2, CXADR, CTSL1, IFN-gamma-R1, IL-18R1, KRT19, KYNU, and TGM2 in a biological sample obtained from the human subject that is lower than a control, and/or (ii) a baseline concentration of at least one protein (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 proteins) selected from the group consisting of PON3, CNTN1, IGFBP3, LEP, Notch 3, TN-R, HSD11B1, FAM19A5, NCAN, F11, GDF-8, CCL28, GALNT10, BCAN, TIMP4, CRISP2, CD207, WNT9A, MBL2, EN-RAGE, TWEAK, CR2, MFAP5, KIT, GH, PFKM, CDSN, CRH, GCP5, KLK6, and DRAXIN in a biological sample obtained from the human subject that is higher than a control.

In some embodiments, the human subject has been previously determined to have (i) a baseline concentration of at least one protein (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 proteins) selected from the group consisting of MCP-3, HAOX1, CA5A, CALCA, IL8, SULT2A1, VAMP5, SPINK1, ENPP7, ACE2, CTSL1, PRSS2, CXCL10, MFGE8, KRT19, ALDH1A1, CES1, REG3A, KYNU, IL-4RA, CDCP1, MVK, FOSB, NFATC3, N2DL-2, DDAH1, IGFBP-1, ALDH3A1, CXADR, PLXNB1, CD74, ENTPD2, PREB, CCL19, HNMT, HTRA2, IL-1RT2, and IL-18R1 in a biological sample obtained from the human subject that is lower than a control, and/or (ii) a baseline concentration of at least one protein (e.g., at least 1, 2, 3, 4, 5, 6, 7, or 8 proteins) selected from the group consisting of PON3, LEP, MBL2, GH, GDF-8, EN-RAGE, CRISP2, and CR2 in a biological sample obtained from the human subject that is higher than a control.

In some embodiments, the human subject has been previously determined to have (i) a baseline concentration of at least one protein (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 proteins) selected from the group consisting of MCP-3, HAOX1, CA5A, CALCA, IL8, SULT2A1, VAMP5, SPINK1, ENPP7, ACE2, CTSL1, PRSS2, CXCL10, MFGE8, KRT19, and ALDH1A1 in a biological sample obtained from the human subject that is lower than a control, and/or (ii) a baseline concentration of at least one protein (e.g., at least 1, 2, or 3 proteins) selected from the group consisting of PON3, LEP, and MBL2 in a biological sample obtained from the human subject that is higher than a control.

In some embodiments, the human subject has been previously determined to have (i) a baseline concentration of at least one protein (e.g., at least 1, 2, 3, 4, 5, 6, 7, or 8 proteins) selected from the group consisting of MCP-3, HAOX1, CA5A, CALCA, IL8, SULT2A1, VAMP5, and SPINK1 in a biological sample obtained from the human subject that is lower than a control, and/or (ii) a baseline concentration of at least one protein (e.g., at least 1 or 2 proteins) selected from the group consisting of PON3 and LEP in a biological sample obtained from the human subject that is higher than a control.

The disclosure also features a method of treating a human subject having, suspected of having, or at risk of developing GvHD, by: providing a biological sample obtained from the human subject; measuring in the biological sample a reduced concentration, as compared to a control, of at least one protein (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 proteins) selected from the group consisting of IL8, HAOX1, ENPP7, ACE2, SULT2A1, MCP-3, CES1, MFGE8, PLXNB1, TNFRSF10A, CCL15, SEMA4C, PREB, NFATC3, CCL19, DLL1, ENTPD2, IL-4RA, EPHA2, FOSB, CXCL10, VAMP5, ALDH3A1, MVK, IL12RB1, CALCA, AHCY, PRSS2, LILRB4, DDAH1, IL-1ra, NECTIN2, PDCD1, CD74, PD-L1, REG3A, CA5A, N2DL-2, CDCP1, U-PAR, SIGLEC7, ANGPTL4, ALDH1A1, SPINK1, HTRA2, PRDX6, IL-1RT2, IGFBP-1, HNMT, TRAIL-R2, CXADR, CTSL1, IFN-gamma-R1, IL-18R1, KRT19, KYNU, and TGM2, and/or an increased concentration, as compared to a control, of at least one protein (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 proteins) selected from the group consisting of PON3, CNTN1, IGFBP3, LEP, Notch 3, TN-R, HSD11B1, FAM19A5, NCAN, F11, GDF-8, CCL28, GALNT10, BCAN, TIMP4, CRISP2, CD207, WNT9A, MBL2, EN-RAGE, TWEAK, CR2, MFAP5, KIT, GH, PFKM, CDSN, CRH, GCP5, KLK6, and DRAXIN; and administering a therapy comprising a JAK inhibitor to the human subject.

In some embodiments, the method includes: measuring in the biological sample a reduced concentration, as compared to a control, of at least one protein (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 proteins) selected from the group consisting of MCP-3, HAOX1, CA5A, CALCA, IL8, SULT2A1, VAMP5, SPINK1, ENPP7, ACE2, CTSL1, PRSS2, CXCL10, MFGE8, KRT19, ALDH1A1, CES1, REG3A, KYNU, IL-4RA, CDCP1, MVK, FOSB, NFATC3, N2DL-2, DDAH1, IGFBP-1, ALDH3A1, CXADR, PLXNB1, CD74, ENTPD2, PREB, CCL19, HNMT, HTRA2, IL-1RT2, and IL-18R1, and/or an increased concentration, as compared to a control, of at least one protein (e.g., at least 1, 2, 3, 4, 5, 6, 7, or 8 proteins) selected from the group consisting of PON3, LEP, MBL2, GH, GDF-8, EN-RAGE, CRISP2, and CR2; and administering the therapy comprising the JAK inhibitor to the human subject.

In some embodiments, the method includes: measuring in the biological sample a reduced concentration, as compared to a control, of at least one protein (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 proteins) selected from the group consisting of MCP-3, HAOX1, CA5A, CALCA, IL8, SULT2A1, VAMP5, SPINK1, ENPP7, ACE2, CTSL1, PRSS2, CXCL10, MFGE8, KRT19, and ALDH1A1, and/or an increased concentration, as compared to a control, of at least one protein (e.g., at least 1, 2, or 3 proteins) selected from the group consisting of PON3, LEP, and MBL2; and administering the therapy comprising the JAK inhibitor to the human subject.

In some embodiments, the method includes: measuring in the biological sample a reduced concentration, as compared to a control, of at least one protein (e.g., at least 1, 2, 3, 4, 5, 6, 7, or 8 proteins) selected from the group consisting of MCP-3, HAOX1, CASA, CALCA, IL8, SULT2A1, VAMP5, and SPINK1, and/or an increased concentration, as compared to a control, of at least one protein (e.g., at least 1 or 2 proteins) selected from the group consisting of PON3 and LEP; and administering the therapy comprising the JAK inhibitor to the human subject.

The disclosure also features a method of predicting the response of a human subject having, suspected of having, or at risk of developing GvHD to a therapy comprising a JAK inhibitor, by: providing a biological sample obtained from the subject before the therapy comprising the JAK inhibitor; and measuring the concentration of at least one protein (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 proteins) selected from the group consisting of PON3, CNTN1, IGFBP3, LEP, Notch 3, TN-R, HSD11B1, FAM19A5, NCAN, F11, GDF-8, CCL28, GALNT10, BCAN, TIMP4, CRISP2, CD207, WNT9A, MBL2, EN-RAGE, TWEAK, CR2, MFAP5, KIT, GH, PFKM, CDSN, CRH, GCP5, KLK6, DRAXIN, IL8, HAOX1, ENPP7, ACE2, SULT2A1, MCP-3, CES1, MFGE8, PLXNB1, TNFRSF10A, CCL15, SEMA4C, PREB, NFATC3, CCL19, DLL1, ENTPD2, IL-4RA, EPHA2, FOSB, CXCL10, VAMP5, ALDH3A1, MVK, IL12RB1, CALCA, AHCY, PRSS2, LILRB4, DDAH1, IL-1ra, NECTIN2, PDCD1, CD74, PD-L1, REG3A, CA5A, N2DL-2, CDCP1, U-PAR, SIGLEC7, ANGPTL4, ALDH1A1, SPINK1, HTRA2, PRDX6, IL-1RT2, IGFBP-1, HNMT, TRAIL-R2, CXADR, CTSL1, IFN-gamma-R1, IL-18R1, KRT19, KYNU, and TGM2 in the biological sample, wherein a reduced concentration, as compared to a control, of at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) of IL8, HAOX1, ENPP7, ACE2, SULT2A1, MCP-3, CES1, MFGE8, PLXNB1, TNFRSF10A, CCL15, SEMA4C, PREB, NFATC3, CCL19, DLL1, ENTPD2, IL-4RA, EPHA2, FOSB, CXCL10, VAMP5, ALDH3A1, MVK, IL12RB1, CALCA, AHCY, PRSS2, LILRB4, DDAH1, IL-1ra, NECTIN2, PDCD1, CD74, PD-L1, REG3A, CA5A, N2DL-2, CDCP1, U-PAR, SIGLEC7, ANGPTL4, ALDH1A1, SPINK1, HTRA2, PRDX6, IL-1RT2, IGFBP-1, HNMT, TRAIL-R2, CXADR, CTSL1, IFN-gamma-R1, IL-18R1, KRT19, KYNU, or TGM2, and/or an increased concentration, as compared to a control, of at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) of PON3, CNTN1, IGFBP3, LEP, Notch 3, TN-R, HSD11B1, FAM19A5, NCAN, F11, GDF-8, CCL28, GALNT10, BCAN, TIMP4, CRISP2, CD207, WNT9A, MBL2, EN-RAGE, TWEAK, CR2, MFAP5, KIT, GH, PFKM, CDSN, CRH, GCP5, KLK6, or DRAXIN is predictive that the subject will respond to the therapy comprising the JAK inhibitor.

In some embodiments, the method includes: measuring the concentration of at least one protein (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 proteins) selected from the group consisting of PON3, LEP, MBL2, GH, GDF-8, EN-RAGE, CRISP2, CR2, MCP-3, HAOX1, CA5A, CALCA, IL8, SULT2A1, VAMP5, SPINK1, ENPP7, ACE2, CTSL1, PRSS2, CXCL10, MFGE8, KRT19, ALDH1A1, CES1, REG3A, KYNU, IL-4RA, CDCP1, MVK, FOSB, NFATC3, N2DL-2, DDAH1, IGFBP-1, ALDH3A1, CXADR, PLXNB1, CD74, ENTPD2, PREB, CCL19, HNMT, HTRA2, IL-1RT2, and IL-18R1 in the biological sample, wherein a reduced concentration, as compared to a control, of at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) of MCP-3, HAOX1, CA5A, CALCA, IL8, SULT2A1, VAMP5, SPINK1, ENPP7, ACE2, CTSL1, PRSS2, CXCL10, MFGE8, KRT19, ALDH1A1, CES1, REG3A, KYNU, IL-4RA, CDCP1, MVK, FOSB, NFATC3, N2DL-2, DDAH1, IGFBP-1, ALDH3A1, CXADR, PLXNB1, CD74, ENTPD2, PREB, CCL19, HNMT, HTRA2, IL-1RT2, or IL-18R1, and/or an increased concentration, as compared to a control, of at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, or 8) of PON3, LEP, MBL2, GH, GDF-8, EN-RAGE, CRISP2, or CR2 is predictive that the subject will respond to the therapy comprising the JAK inhibitor.

In some embodiments, the method includes: measuring the concentration of at least one protein (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 proteins) selected from the group consisting of PON3, LEP, MBL2, MCP-3, HAOX1, CA5A, CALCA, IL8, SULT2A1, VAMP5, SPINK1, ENPP7, ACE2, CTSL1, PRSS2, CXCL10, MFGE8, KRT19, and ALDH1A1 in the biological sample, wherein a reduced concentration, as compared to a control, of at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16) of MCP-3, HAOX1, CA5A, CALCA, IL8, SULT2A1, VAMP5, SPINK1, ENPP7, ACE2, CTSL1, PRSS2, CXCL10, MFGE8, KRT19, or ALDH1A1, and/or an increased concentration, as compared to a control, of at least one (e.g., at least 1, 2, or 3) of PON3, LEP, or MBL2 is predictive that the subject will respond to the therapy comprising the JAK inhibitor.

In some embodiments, the method includes: measuring the concentration of at least one protein (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 proteins) selected from the group consisting of PON3, LEP, MCP-3, HAOX1, CA5A, CALCA, IL8, SULT2A1, VAMP5, and SPINK1 in the biological sample, wherein a reduced concentration, as compared to a control, of at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, or 8) of MCP-3, HAOX1, CA5A, CALCA, IL8, SULT2A1, VAMP5, or SPINK1, and/or an increased concentration, as compared to a control, of at least one (e.g., at least 1 or 2) of PON3 or LEP is predictive that the subject will respond to the therapy comprising the JAK inhibitor.

In some embodiments of the methods described herein, the control is a pre-established cut-off value.

In some embodiments of the methods described herein, the control is the concentration of the protein in a sample or samples obtained from one or more subjects that have not responded to treatment with the JAK inhibitor.

The disclosure also features a method for measuring the amount of a protein in a sample, by: providing a biological sample obtained from a human subject having, suspected of having, or at risk of developing GvHD; and measuring the concentration of at least one protein (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 proteins) selected from the group consisting of PON3, CNTN1, IGFBP3, LEP, Notch 3, TN-R, HSD11B1, FAM19A5, NCAN, F11, GDF-8, CCL28, GALNT10, BCAN, TIMP4, CRISP2, CD207, WNT9A, MBL2, EN-RAGE, TWEAK, CR2, MFAP5, KIT, GH, PFKM, CDSN, CRH, GCP5, KLK6, DRAXIN, IL8, HAOX1, ENPP7, ACE2, SULT2A1, MCP-3, CES1, MFGE8, PLXNB1, TNFRSF10A, CCL15, SEMA4C, PREB, NFATC3, CCL19, DLL1, ENTPD2, IL-4RA, EPHA2, FOSB, CXCL10, VAMP5, ALDH3A1, MVK, IL12RB1, CALCA, AHCY, PRSS2, LILRB4, DDAH1, IL-1ra, NECTIN2, PDCD1, CD74, PD-L1, REG3A, CA5A, N2DL-2, CDCP1, U-PAR, SIGLEC7, ANGPTL4, ALDH1A1, SPINK1, HTRA2, PRDX6, IL-1RT2, IGFBP-1, HNMT, TRAIL-R2, CXADR, CTSL1, IFN-gamma-R1, IL-18R1, KRT19, KYNU, and TGM2 in the biological sample.

In some embodiments, the method includes measuring the concentration of at least one protein (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 proteins) selected from the group consisting of PON3, LEP, MBL2, GH, GDF-8, EN-RAGE, CRISP2, CR2, MCP-3, HAOX1, CA5A, CALCA, IL8, SULT2A1, VAMP5, SPINK1, ENPP7, ACE2, CTSL1, PRSS2, CXCL10, MFGE8, KRT19, ALDH1A1, CES1, REG3A, KYNU, IL-4RA, CDCP1, MVK, FOSB, NFATC3, N2DL-2, DDAH1, IGFBP-1, ALDH3A1, CXADR, PLXNB1, CD74, ENTPD2, PREB, CCL19, HNMT, HTRA2, IL-1RT2, and IL-18R1 in the biological sample.

In some embodiments, the method includes measuring the concentration of at least one protein (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 proteins) selected from the group consisting of PON3, LEP, MBL2, MCP-3, HAOX1, CA5A, CALCA, IL8, SULT2A1, VAMP5, SPINK1, ENPP7, ACE2, CTSL1, PRSS2, CXCL10, MFGE8, KRT19, and ALDH1A1 in the biological sample.

In some embodiments, the method includes measuring the concentration of at least one protein (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 proteins) selected from the group consisting of PON3, LEP, MCP-3, HAOX1, CA5A, CALCA, IL8, SULT2A1, VAMP5, and SPINK1 in the biological sample.

In some embodiments of the methods described herein, the concentrations of no more than 50, 40, 30, 20, 15, 10, or 5 proteins are measured.

The disclosure also features a method of treating a human subject having, suspected of having, or at risk of developing GvHD, by: measuring, in a first biological sample obtained from the human subject prior to administering a therapy comprising a JAK inhibitor, the concentration of at least one protein (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 proteins) selected from the group consisting of INPPL1, LAT2, CLEC7A, PPP1R9B, NEMO, SH2B3, BCR, CD5, DNAJB1, CCL17, ITGB2, BANK1, TPSAB1, YES1, LAMP3, GM-CSF-R-alpha, CNTNAP2, ZBTB16, CD163, TXLNA, MEPE, BACH1, MAX, NFKBIE, hOSCAR, LAT, PTPRJ, SIRT2, SIRPB1, AXIN1, EIF4G1, PTX3, TRIM5, IDUA, NCF2, SELP, ARHGEF12, CASP-3, CD27, MAP4K5, DAPP1, PRDX5, TLT-2, PARK7, IL2-RA, FOXO1, ST1A1, GRAP2, NBN, CD93, FCGR2A, DCTN1, IRF9, HAVCR2, CD84, STX8, LY9, ZBTB16, CD200R1, TOP2B, THY 1, PRKRA, ITGB1BP2, CD48, CD244, HCLS1, MPO, SIT1, ICAM3, SOST, DDX58, TNF-R2, TRAF2, SMAD1, LAIR-2, PIK3AP1, VSIG4, SIGLEC10, CD6, SKAP1, FCRL5, CD177, KLRD1, ERBB2IP, MILR1, MIF, SNAP23, NUB1, TIGAR, STAMPB, DSC2, LAIR1, FKBP1B, RASSF2, FATC1, CBL, IgG Fc receptor II-b, GLO1, PVALB, SCAMP3, SLAMF8, STX16, TNF-R1, DFFA, PPP1R2, ANG-1, CCL5, MAP2K6, CRKL, CD38, CXCL5, PILRA, IRAK1, CA13, STX6, PRTN3, IL-5R-alpha, ESM-1, EGLN1, CLEC1B, TYMP, SNAP29, PDGF subunit A, TNFRSF11A, gal-8, GCNT1, STK4, TNC, THBS4, CLEC4D, SIGLEC6, WASF1, WAS, COMT, RETN, SH2D1A, RNASE3, PAR-1, CD69, SIGLEC1, FR-gamma, ADAM 8, AZU1, AREG, SDC4, DCTN2, BID, RELT, CLEC5A, APEX1, PSP-D, FGR, SELE, SELL, MESDC2, IQGAP2, CRTAM, LILRB2, TANK, CPXM1, ARSB, SLAMF1, PEBP1, STIP1, PDGF subunit B, SCARF1, DEFA1, EPHB4, ARHGAP1, CLM-1, DAB2, LYN, CASP-8, APBB1IP, ANXA11, ICAM1, PRKCQ, VCAM1, HDGF, CD2AP, TNFRSF6B, CLEC1A, TNFRSF14, TACC3, MMP-1, NRP1, ZBTB17, NADK, PLXNA4, MMP-9, NCR1, AMIGO2, FES, CD79B, TNXB, TXNDC5, TRANCE, ARG1, PCDH17, LRMP, C1QTNF1, CLM-6, CKAP4, APP, PGLYRP1, LILRA5, CLEC10A, NMNAT1, IL-6RA, ATG4A, TIMP1, COCH, DKN1A, CD1C, DECR1, DAG1, IGFBP-2, RET, GSAP, PILRB, CLEC6A, PECAM-1, PXN, ADGRG1, DPP7, TDRKH, Siglec-9, CD40-L, VEGFC, LYVE1, FADD, FCRL1, EGF, HGF, GZMH, CLEC4G, LY75, PRDX3, COL4A1, CEACAM8, SEMA7A, NUDT5, FCRL6, PAPPA, FASLG, GRN, MATN3, TMPRSS15, CCL11, FAM3B, MMP7, NCAM1, Gal-3, CCL25, THPO, hK14, KIM1, Flt3L, PLIN1, SPON2, Gal-4, FABP4, DNER, GAL, CPM, VWC2, PPY, PAM, PVR, SERPINA5, ST3GAL1, CST5, CES2, CNDP1, CX3CL1, HO-1, PRELP, ADM, VSIG2, FABP2, CEACAM5, SLITRK2, MCP-1, NTRK3, CLUL1, CXCL16, SCF, TMPRSS5, REG4, hK11, SCGB3A1, DKKL1, NEP, CPA2, Ep-CAM, THBS2, GPNMB, ITGB5, GT, APLP1, TACSTD2, NINJ1, REN, GCG, SERPINA9, KAZALD1, SERPINA12, PODXL, AMN, IGF1R, LTBP2, ANGPTL3, SCARA5, B4GAT1, ROBO2, PDGFC, CA12, DDC, EDIL3, XPNPEP2, PRTG, NQO2, AMBP, ERBB2, IL6, MCP-1, VEGFD, GDF-2, MUC-16, KLK10, FAM3C, uPA, AGR2, METRNL, RTN4R, IGF2R, NTRK2, ITGB6, SCARF2, SCGB3A2, RGMB, EZR, PROC, FURIN, PIgR, and SMOC2; administering the therapy comprising the JAK inhibitor to the human subject; and measuring, in a second biological sample obtained from the human subject after administering the therapy comprising the JAK inhibitor, a reduced concentration, as compared to the first biological sample, of at least one protein (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 proteins) selected from the group consisting of INPPL1, LAT2, CLEC7A, PPP1R9B, NEMO, SH2B3, BCR, CD5, DNAJB1, CCL17, ITGB2, BANK1, TPSAB1, YES1, LAMP3, GM-CSF-R-alpha, CNTNAP2, ZBTB16, CD163, TXLNA, MEPE, BACH1, MAX, NFKBIE, hOSCAR, LAT, PTPRJ, SIRT2, SIRPB1, AXIN1, EIF4G1, PTX3, TRIM5, IDUA, NCF2, SELP, ARHGEF12, CASP-3, CD27, MAP4K5, DAPP1, PRDX5, TLT-2, PARK7, IL2-RA, FOXO1, ST1A1, GRAP2, NBN, CD93, FCGR2A, DCTN1, IRF9, HAVCR2, CD84, STX8, LY9, ZBTB16, CD200R1, TOP2B, THY 1, PRKRA, ITGB1BP2, CD48, CD244, HCLS1, MPO, SIT1, ICAM3, SOST, DDX58, TNF-R2, TRAF2, SMAD1, LAIR-2, PIK3AP1, VSIG4, SIGLEC10, CD6, SKAP1, FCRL5, CD177, KLRD1, ERBB2IP, MILR1, MIF, SNAP23, NUB1, TIGAR, STAMPB, DSC2, LAIR1, FKBP1B, RASSF2, FATC1, CBL, IgG Fc receptor II-b, GLO1, PVALB, SCAMP3, SLAMF8, STX16, TNF-R1, DFFA, PPP1R2, ANG-1, CCL5, MAP2K6, CRKL, CD38, CXCL5, PILRA, IRAK1, CA13, STX6, PRTN3, IL-5R-alpha, ESM-1, EGLN1, CLEC1B, TYMP, SNAP29, PDGF subunit A, TNFRSF11A, gal-8, GCNT1, STK4, TNC, THBS4, CLEC4D, SIGLEC6, WASF1, WAS, COMT, RETN, SH2D1A, RNASE3, PAR-1, CD69, SIGLEC1, FR-gamma, ADAM 8, AZU1, AREG, SDC4, DCTN2, BID, RELT, CLEC5A, APEX1, PSP-D, FGR, SELE, SELL, MESDC2, IQGAP2, CRTAM, LILRB2, TANK, CPXM1, ARSB, SLAMF1, PEBP1, STIP1, PDGF subunit B, SCARF1, DEFA1, EPHB4, ARHGAP1, CLM-1, DAB2, LYN, CASP-8, APBB1IP, ANXA11, ICAM1, PRKCQ, VCAM1, HDGF, CD2AP, TNFRSF6B, CLEC1A, TNFRSF14, TACC3, MMP-1, NRP1, ZBTB17, NADK, PLXNA4, MMP-9, NCR1, AMIGO2, FES, CD79B, TNXB, TXNDC5, TRANCE, ARG1, PCDH17, LRMP, C1QTNF1, CLM-6, CKAP4, APP, PGLYRP1, LILRA5, CLEC10A, NMNAT1, IL-6RA, ATG4A, TIMP1, COCH, DKN1A, CD1C, DECR1, DAG1, IGFBP-2, RET, GSAP, PILRB, CLEC6A, PECAM-1, PXN, ADGRG1, DPP7, TDRKH, Siglec-9, CD40-L, VEGFC, LYVE1, FADD, FCRL1, EGF, HGF, GZMH, CLEC4G, LY75, PRDX3, COL4A1, CEACAM8, SEMA7A, NUDT5, FCRL6, PAPPA, FASLG, GRN, and MATN3, and/or an increased concentration, as compared to the first biological sample, of at least one protein (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 proteins) selected from the group consisting of TMPRSS15, CCL11, FAM3B, MMP7, NCAM1, Gal-3, CCL25, THPO, hK14, KIM1, Flt3L, PLIN1, SPON2, Gal-4, FABP4, DNER, GAL, CPM, VWC2, PPY, PAM, PVR, SERPINA5, ST3GAL1, CST5, CES2, CNDP1, CX3CL1, HO-1, PRELP, ADM, VSIG2, FABP2, CEACAM5, SLITRK2, MCP-1, NTRK3, CLUL1, CXCL16, SCF, TMPRSS5, REG4, hK11, SCGB3A1, DKKL1, NEP, CPA2, Ep-CAM, THBS2, GPNMB, ITGB5, GT, APLP1, TACSTD2, NINE, REN, GCG, SERPINA9, KAZALD1, SERPINA12, PODXL, AMN, IGF1R, LTBP2, ANGPTL3, SCARA5, B4GAT1, ROBO2, PDGFC, CA12, DDC, EDIL3, XPNPEP2, PRTG, NQO2, AMBP, ERBB2, IL6, MCP-1, VEGFD, GDF-2, MUC-16, KLK10, FAM3C, uPA, AGR2, METRNL, RTN4R, IGF2R, NTRK2, ITGB6, SCARF2, SCGB3A2, RGMB, EZR, PROC, FURIN, PIgR, and SMOC2.

In some embodiments, the method includes: measuring, in the first biological sample obtained from the human subject prior to administering the therapy comprising the JAK inhibitor, the concentration of at least one protein (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 proteins) selected from the group consisting of TMPRSS15, CCL11, FAM3B, MMP7, NCAM1, Gal-3, CCL25, THPO, hK14, KIM1, Flt3L, PLIN1, SPON2, Gal-4, FABP4, DNER, GAL, CPM, VWC2, PPY, PAM, PVR, SERPINA5, ST3GAL1, CST5, CES2, CNDP1, CX3CL1, HO-1, PRELP, ADM, VSIG2, FABP2, CEACAM5, SLITRK2, MCP-1, NTRK3, CLUL1, CXCL16, SCF, TMPRSS5, REG4, hK11, SCGB3A1, DKKL1, NEP, CPA2, Ep-CAM, THBS2, GPNMB, INPPL1, LAT2, CLEC7A, PPP1R9B, NEMO, SH2B3, BCR, CD5, DNAJB1, CCL17, ITGB2, BANK1, TPSAB1, YES1, LAMP3, GM-CSF-R-alpha, CNTNAP2, ZBTB16, CD163, TXLNA, MEPE, BACH1, MAX, NFKBIE, hOSCAR, LAT, PTPRJ, SIRT2, SIRPB1, AXIN1, EIF4G1, PTX3, TRIM5, IDUA, NCF2, SELP, ARHGEF12, CASP-3, CD27, MAP4K5, DAPP1, PRDX5, TLT-2, PARK7, IL2-RA, FOXO1, ST1A1, GRAP2, NBN, CD93, FCGR2A, DCTN1, IRF9, HAVCR2, CD84, STX8, LY9, ZBTB16, CD200R1, TOP2B, THY 1, PRKRA, ITGB1BP2, CD48, CD244, HCLS1, MPO, SIT1, ICAM3, SOST, DDX58, TNF-R2, TRAF2, SMAD1, LAIR-2, PIK3AP1, VSIG4, SIGLEC10, CD6, SKAP1, FCRL5, CD177, KLRD1, ERBB2IP, MILR1, MIF, SNAP23, NUB1, TIGAR, STAMPB, DSC2, LAIR1, FKBP1B, RASSF2, FATC1, CBL, IgG Fc receptor II-b, GLO1, PVALB, SCAMP3, SLAMF8, STX16, TNF-R1, DFFA, PPP1R2, ANG-1, CCL5, MAP2K6, CRKL, CD38, CXCL5, PILRA, IRAK1, CA13, STX6, PRTN3, IL-5R-alpha, ESM-1, EGLN1, CLEC1B, TYMP, SNAP29, PDGF subunit A, TNFRSF11A, gal-8, GCNT1, STK4, TNC, THBS4, CLEC4D, SIGLEC6, WASF1, WAS, COMT, RETN, SH2D1A, RNASE3, PAR-1, CD69, SIGLEC1, FR-gamma, ADAM 8, AZU1, AREG, SDC4, DCTN2, BID, and RELT; administering the therapy comprising the JAK inhibitor to the human subject; and measuring, in the second biological sample obtained from the human subject after administering the therapy comprising the JAK inhibitor, a reduced concentration, as compared to the first biological sample, of at least one protein (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 proteins) selected from the group consisting of INPPL1, LAT2, CLEC7A, PPP1R9B, NEMO, SH2B3, BCR, CD5, DNAJB1, CCL17, ITGB2, BANK1, TPSAB1, YES1, LAMP3, GM-CSF-R-alpha, CNTNAP2, ZBTB16, CD163, TXLNA, MEPE, BACH1, MAX, NFKBIE, hOSCAR, LAT, PTPRJ, SIRT2, SIRPB1, AXIN1, EIF4G1, PTX3, TRIM5, IDUA, NCF2, SELP, ARHGEF12, CASP-3, CD27, MAP4K5, DAPP1, PRDX5, TLT-2, PARK7, IL2-RA, FOXO1, ST1A1, GRAP2, NBN, CD93, FCGR2A, DCTN1, IRF9, HAVCR2, CD84, STX8, LY9, ZBTB16, CD200R1, TOP2B, THY 1, PRKRA, ITGB1BP2, CD48, CD244, HCLS1, MPO, SIT1, ICAM3, SOST, DDX58, TNF-R2, TRAF2, SMAD1, LAIR-2, PIK3AP1, VSIG4, SIGLEC10, CD6, SKAP1, FCRL5, CD177, KLRD1, ERBB2IP, MILR1, MIF, SNAP23, NUB1, TIGAR, STAMPB, DSC2, LAIR1, FKBP1B, RASSF2, FATC1, CBL, IgG Fc receptor II-b, GLO1, PVALB, SCAMP3, SLAMF8, STX16, TNF-R1, DFFA, PPP1R2, ANG-1, CCL5, MAP2K6, CRKL, CD38, CXCL5, PILRA, IRAK1, CA13, STX6, PRTN3, IL-5R-alpha, ESM-1, EGLN1, CLEC1B, TYMP, SNAP29, PDGF subunit A, TNFRSF11A, gal-8, GCNT1, STK4, TNC, THBS4, CLEC4D, SIGLEC6, WASF1, WAS, COMT, RETN, SH2D1A, RNASE3, PAR-1, CD69, SIGLEC1, FR-gamma, ADAM 8, AZU1, AREG, SDC4, DCTN2, BID, and RELT, and/or an increased concentration, as compared to the first biological sample, of at least one protein (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 proteins) selected from the group consisting of TMPRSS15, CCL11, FAM3B, MMP7, NCAM1, Gal-3, CCL25, THPO, hK14, KIM1, Flt3L, PLIN1, SPON2, Gal-4, FABP4, DNER, GAL, CPM, VWC2, PPY, PAM, PVR, SERPINA5, ST3GAL1, CST5, CES2, CNDP1, CX3CL1, HO-1, PRELP, ADM, VSIG2, FABP2, CEACAM5, SLITRK2, MCP-1, NTRK3, CLUL1, CXCL16, SCF, TMPRSS5, REG4, hK11, SCGB3A1, DKKL1, NEP, CPA2, Ep-CAM, THBS2, and GPNMB.

In some embodiments, the method includes: measuring, in the first biological sample obtained from the human subject prior to administering the therapy comprising the JAK inhibitor, the concentration of at least one protein (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 proteins) selected from the group consisting of TMPRSS15, CCL11, FAM3B, MMP7, NCAM1, Gal-3, CCL25, THPO, hK14, KIM1, INPPL1, LAT2, CLEC7A, PPP1R9B, NEMO, SH2B3, BCR, CD5, DNAJB1, CCL17, ITGB2, BANK1, TPSAB1, YES1, LAMP3, GM-CSF-R-alpha, CNTNAP2, ZBTB16, CD163, TXLNA, MEPE, BACH1, MAX, NFKBIE, hOSCAR, LAT, PTPRJ, SIRT2, SIRPB1, AXIN1, EIF4G1, PTX3, TRIM5, IDUA, NCF2, SELP, ARHGEF12, CASP-3, CD27, MAP4K5, DAPP1, PRDX5, TLT-2, PARK7, IL2-RA, FOXO1, ST1A1, GRAP2, NBN, CD93, FCGR2A, DCTN1, IRF9, and HAVCR2; administering the therapy comprising the JAK inhibitor to the human subject; and measuring, in the second biological sample obtained from the human subject after administering the therapy comprising the JAK inhibitor, a reduced concentration, as compared to the first biological sample, of at least one protein (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 proteins) selected from the group consisting of INPPL1, LAT2, CLEC7A, PPP1R9B, NEMO, SH2B3, BCR, CD5, DNAJB1, CCL17, ITGB2, BANK1, TPSAB1, YES1, LAMP3, GM-CSF-R-alpha, CNTNAP2, ZBTB16, CD163, TXLNA, MEPE, BACH1, MAX, NFKBIE, hOSCAR, LAT, PTPRJ, SIRT2, SIRPB1, AXIN1, EIF4G1, PTX3, TRIM5, IDUA, NCF2, SELP, ARHGEF12, CASP-3, CD27, MAP4K5, DAPP1, PRDX5, TLT-2, PARK7, IL2-RA, FOXO1, ST1A1, GRAP2, NBN, CD93, FCGR2A, DCTN1, IRF9, and HAVCR2, and/or an increased concentration, as compared to the first biological sample, of at least one protein (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 proteins) selected from the group consisting of TMPRSS15, CCL11, FAM3B, MMP7, NCAM1, Gal-3, CCL25, THPO, hK14, and KIM1.

In some embodiments, the method includes: measuring, in the first biological sample obtained from the human subject prior to administering the therapy comprising the JAK inhibitor, the concentration of at least one protein (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 proteins) selected from the group consisting of TMPRSS15, CCL11, FAM3B, MMP7, NCAM1, Gal-3, CCL25, INPPL1, LAT2, CLEC7A, PPP1R9B, NEMO, SH2B3, BCR, CD5, DNAJB1, CCL17, ITGB2, BANK1, TPSAB1, YES1, LAMP3, GM-CSF-R-alpha, and CNTNAP2; administering the therapy comprising the JAK inhibitor to the human subject; and measuring, in the second biological sample obtained from the human subject after administering the therapy comprising the JAK inhibitor, a reduced concentration, as compared to the first biological sample, of at least one protein (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 proteins) selected from the group consisting of INPPL1, LAT2, CLEC7A, PPP1R9B, NEMO, SH2B3, BCR, CD5, DNAJB1, CCL17, ITGB2, BANK1, TPSAB1, YES1, LAMP3, GM-CSF-R-alpha, and CNTNAP2, and/or an increased concentration, as compared to the first biological sample, of at least one protein (e.g., at least 1, 2, 3, 4, 5, 6, or 7 proteins) selected from the group consisting of TMPRSS15, CCL11, FAM3B, MMP7, NCAM1, Gal-3, and CCL25.

In some embodiments, the method includes: measuring, in the first biological sample obtained from the human subject prior to administering the therapy comprising the JAK inhibitor, the concentration of at least one protein (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 proteins) selected from the group consisting of TMPRSS15, CCL11, FAM3B, MMP7, NCAM1, INPPL1, LAT2, CLEC7A, PPP1R9B, NEMO, SH2B3, BCR, CD5, DNAJB1, CCL17, and ITGB2; administering the therapy comprising the JAK inhibitor to the human subject; and measuring, in the second biological sample obtained from the human subject after administering the therapy comprising the JAK inhibitor, a reduced concentration, as compared to the first biological sample, of at least one protein (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 proteins) selected from the group consisting of INPPL1, LAT2, CLEC7A, PPP1R9B, NEMO, SH2B3, BCR, CD5, DNAJB1, CCL17, and ITGB2, and/or an increased concentration, as compared to the first biological sample, of at least one protein (e.g., at least 1, 2, 3, 4, or 5 proteins) selected from the group consisting of TMPRSS15, CCL11, FAM3B, MMP7, and NCAM1.

The disclosure also features a method of identifying a therapeutic response of a human subject having, suspected of having, or at risk of developing GvHD to a therapy comprising a JAK inhibitor, by: providing a first biological sample obtained from the human subject before administering the therapy comprising the JAK inhibitor; measuring the concentration of at least one protein (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 proteins) selected from the group consisting of INPPL1, LAT2, CLEC7A, PPP1R9B, NEMO, SH2B3, BCR, CD5, DNAJB1, CCL17, ITGB2, BANK1, TPSAB1, YES1, LAMP3, GM-CSF-R-alpha, CNTNAP2, ZBTB16, CD163, TXLNA, MEPE, BACH1, MAX, NFKBIE, hOSCAR, LAT, PTPRJ, SIRT2, SIRPB1, AXIN1, EIF4G1, PTX3, TRIM5, IDUA, NCF2, SELP, ARHGEF12, CASP-3, CD27, MAP4K5, DAPP1, PRDX5, TLT-2, PARK7, IL2-RA, FOXO1, ST1A1, GRAP2, NBN, CD93, FCGR2A, DCTN1, IRF9, HAVCR2, CD84, STX8, LY9, ZBTB16, CD200R1, TOP2B, THY 1, PRKRA, ITGB1BP2, CD48, CD244, HCLS1, MPO, SIT1, ICAM3, SOST, DDX58, TNF-R2, TRAF2, SMAD1, LAIR-2, PIK3AP1, VSIG4, SIGLEC10, CD6, SKAP1, FCRL5, CD177, KLRD1, ERBB2IP, MILR1, MIF, SNAP23, NUB1, TIGAR, STAMPB, DSC2, LAIR1, FKBP1B, RASSF2, FATC1, CBL, IgG Fc receptor II-b, GLO1, PVALB, SCAMP3, SLAMF8, STX16, TNF-R1, DFFA, PPP1R2, ANG-1, CCL5, MAP2K6, CRKL, CD38, CXCL5, PILRA, IRAK1, CA13, STX6, PRTN3, IL-5R-alpha, ESM-1, EGLN1, CLEC1B, TYMP, SNAP29, PDGF subunit A, TNFRSF11A, gal-8, GCNT1, STK4, TNC, THBS4, CLEC4D, SIGLEC6, WASF1, WAS, COMT, RETN, SH2D1A, RNASE3, PAR-1, CD69, SIGLEC1, FR-gamma, ADAM 8, AZU1, AREG, SDC4, DCTN2, BID, RELT, CLEC5A, APEX1, PSP-D, FGR, SELE, SELL, MESDC2, IQGAP2, CRTAM, LILRB2, TANK, CPXM1, ARSB, SLAMF1, PEBP1, STIP1, PDGF subunit B, SCARF1, DEFA1, EPHB4, ARHGAP1, CLM-1, DAB2, LYN, CASP-8, APBB1IP, ANXA11, ICAM1, PRKCQ, VCAM1, HDGF, CD2AP, TNFRSF6B, CLEC1A, TNFRSF14, TACC3, MMP-1, NRP1, ZBTB17, NADK, PLXNA4, MMP-9, NCR1, AMIGO2, FES, CD79B, TNXB, TXNDC5, TRANCE, ARG1, PCDH17, LRMP, C1QTNF1, CLM-6, CKAP4, APP, PGLYRP1, LILRA5, CLEC10A, NMNAT1, IL-6RA, ATG4A, TIMP1, COCH, DKN1A, CD1C, DECR1, DAG1, IGFBP-2, RET, GSAP, PILRB, CLEC6A, PECAM-1, PXN, ADGRG1, DPP7, TDRKH, Siglec-9, CD40-L, VEGFC, LYVE1, FADD, FCRL1, EGF, HGF, GZMH, CLEC4G, LY75, PRDX3, COL4A1, CEACAM8, SEMA7A, NUDT5, FCRL6, PAPPA, FASLG, GRN, MATN3, TMPRSS15, CCL11, FAM3B, MMP7, NCAM1, Gal-3, CCL25, THPO, hK14, KIM1, Flt3L, PLIN1, SPON2, Gal-4, FABP4, DNER, GAL, CPM, VWC2, PPY, PAM, PVR, SERPINA5, ST3GAL1, CST5, CES2, CNDP1, CX3CL1, HO-1, PRELP, ADM, VSIG2, FABP2, CEACAM5, SLITRK2, MCP-1, NTRK3, CLUL1, CXCL16, SCF, TMPRSS5, REG4, hK11, SCGB3A1, DKKL1, NEP, CPA2, Ep-CAM, THBS2, GPNMB, ITGB5, GT, APLP1, TACSTD2, NINJ1, REN, GCG, SERPINA9, KAZALD1, SERPINA12, PODXL, AMN, IGF1R, LTBP2, ANGPTL3, SCARA5, B4GAT1, ROBO2, PDGFC, CA12, DDC, EDIL3, XPNPEP2, PRTG, NQO2, AMBP, ERBB2, IL6, MCP-1, VEGFD, GDF-2, MUC-16, KLK10, FAM3C, uPA, AGR2, METRNL, RTN4R, IGF2R, NTRK2, ITGB6, SCARF2, SCGB3A2, RGMB, EZR, PROC, FURIN, PIgR, and SMOC2 in the first biological sample; providing a second biological sample obtained from the subject after administering the therapy comprising the JAK inhibitor; and measuring the concentration of at least one protein (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 proteins) selected from the group consisting of INPPL1, LAT2, CLEC7A, PPP1R9B, NEMO, SH2B3, BCR, CD5, DNAJB1, CCL17, ITGB2, BANK1, TPSAB1, YES1, LAMP3, GM-CSF-R-alpha, CNTNAP2, ZBTB16, CD163, TXLNA, MEPE, BACH1, MAX, NFKBIE, hOSCAR, LAT, PTPRJ, SIRT2, SIRPB1, AXIN1, EIF4G1, PTX3, TRIM5, IDUA, NCF2, SELP, ARHGEF12, CASP-3, CD27, MAP4K5, DAPP1, PRDX5, TLT-2, PARK7, IL2-RA, FOXO1, ST1A1, GRAP2, NBN, CD93, FCGR2A, DCTN1, IRF9, HAVCR2, CD84, STX8, LY9, ZBTB16, CD200R1, TOP2B, THY 1, PRKRA, ITGB1BP2, CD48, CD244, HCLS1, MPO, SIT1, ICAM3, SOST, DDX58, TNF-R2, TRAF2, SMAD1, LAIR-2, PIK3AP1, VSIG4, SIGLEC10, CD6, SKAP1, FCRL5, CD177, KLRD1, ERBB2IP, MILR1, MIF, SNAP23, NUB1, TIGAR, STAMPB, DSC2, LAIR1, FKBP1B, RASSF2, FATC1, CBL, IgG Fc receptor II-b, GLO1, PVALB, SCAMP3, SLAMF8, STX16, TNF-R1, DFFA, PPP1R2, ANG-1, CCL5, MAP2K6, CRKL, CD38, CXCL5, PILRA, IRAK1, CA13, STX6, PRTN3, IL-5R-alpha, ESM-1, EGLN1, CLEC1B, TYMP, SNAP29, PDGF subunit A, TNFRSF11A, gal-8, GCNT1, STK4, TNC, THBS4, CLEC4D, SIGLEC6, WASF1, WAS, COMT, RETN, SH2D1A, RNASE3, PAR-1, CD69, SIGLEC1, FR-gamma, ADAM 8, AZU1, AREG, SDC4, DCTN2, BID, RELT, CLEC5A, APEX1, PSP-D, FGR, SELE, SELL, MESDC2, IQGAP2, CRTAM, LILRB2, TANK, CPXM1, ARSB, SLAMF1, PEBP1, STIP1, PDGF subunit B, SCARF1, DEFA1, EPHB4, ARHGAP1, CLM-1, DAB2, LYN, CASP-8, APBB1IP, ANXA11, ICAM1, PRKCQ, VCAM1, HDGF, CD2AP, TNFRSF6B, CLEC1A, TNFRSF14, TACC3, MMP-1, NRP1, ZBTB17, NADK, PLXNA4, MMP-9, NCR1, AMIGO2, FES, CD79B, TNXB, TXNDC5, TRANCE, ARG1, PCDH17, LRMP, C1QTNF1, CLM-6, CKAP4, APP, PGLYRP1, LILRA5, CLEC10A, NMNAT1, IL-6RA, ATG4A, TIMP1, COCH, DKN1A, CD1C, DECR1, DAG1, IGFBP-2, RET, GSAP, PILRB, CLEC6A, PECAM-1, PXN, ADGRG1, DPP7, TDRKH, Siglec-9, CD40-L, VEGFC, LYVE1, FADD, FCRL1, EGF, HGF, GZMH, CLEC4G, LY75, PRDX3, COL4A1, CEACAM8, SEMA7A, NUDT5, FCRL6, PAPPA, FASLG, GRN, MATN3, TMPRSS15, CCL11, FAM3B, MMP7, NCAM1, Gal-3, CCL25, THPO, hK14, KIM1, Flt3L, PLIN1, SPON2, Gal-4, FABP4, DNER, GAL, CPM, VWC2, PPY, PAM, PVR, SERPINA5, ST3GAL1, CST5, CES2, CNDP1, CX3CL1, HO-1, PRELP, ADM, VSIG2, FABP2, CEACAM5, SLITRK2, MCP-1, NTRK3, CLUL1, CXCL16, SCF, TMPRSS5, REG4, hK11, SCGB3A1, DKKL1, NEP, CPA2, Ep-CAM, THBS2, GPNMB, ITGB5, GT, APLP1, TACSTD2, NINJ1, REN, GCG, SERPINA9, KAZALD1, SERPINA12, PODXL, AMN, IGF1R, LTBP2, ANGPTL3, SCARA5, B4GAT1, ROBO2, PDGFC, CA12, DDC, EDIL3, XPNPEP2, PRTG, NQO2, AMBP, ERBB2, IL6, MCP-1, VEGFD, GDF-2, MUC-16, KLK10, FAM3C, uPA, AGR2, METRNL, RTN4R, IGF2R, NTRK2, ITGB6, SCARF2, SCGB3A2, RGMB, EZR, PROC, FURIN, PIgR, and SMOC2 in the second biological sample, wherein a reduced concentration in the second biological sample, as compared to the first biological sample, of at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) of INPPL1, LAT2, CLEC7A, PPP1R9B, NEMO, SH2B3, BCR, CD5, DNAJB1, CCL17, ITGB2, BANK1, TPSAB1, YES1, LAMP3, GM-CSF-R-alpha, CNTNAP2, ZBTB16, CD163, TXLNA, MEPE, BACH1, MAX, NFKBIE, hOSCAR, LAT, PTPRJ, SIRT2, SIRPB1, AXIN1, EIF4G1, PTX3, TRIM5, IDUA, NCF2, SELP, ARHGEF12, CASP-3, CD27, MAP4K5, DAPP1, PRDX5, TLT-2, PARK7, IL2-RA, FOXO1, ST1A1, GRAP2, NBN, CD93, FCGR2A, DCTN1, IRF9, HAVCR2, CD84, STX8, LY9, ZBTB16, CD200R1, TOP2B, THY 1, PRKRA, ITGB1BP2, CD48, CD244, HCLS1, MPO, SIT1, ICAM3, SOST, DDX58, TNF-R2, TRAF2, SMAD1, LAIR-2, PIK3AP1, VSIG4, SIGLEC10, CD6, SKAP1, FCRL5, CD177, KLRD1, ERBB2IP, MILR1, MIF, SNAP23, NUB1, TIGAR, STAMPB, DSC2, LAIR1, FKBP1B, RASSF2, FATC1, CBL, IgG Fc receptor II-b, GLO1, PVALB, SCAMP3, SLAMF8, STX16, TNF-R1, DFFA, PPP1R2, ANG-1, CCL5, MAP2K6, CRKL, CD38, CXCL5, PILRA, IRAK1, CA13, STX6, PRTN3, IL-5R-alpha, ESM-1, EGLN1, CLEC1B, TYMP, SNAP29, PDGF subunit A, TNFRSF11A, gal-8, GCNT1, STK4, TNC, THBS4, CLEC4D, SIGLEC6, WASF1, WAS, COMT, RETN, SH2D1A, RNASE3, PAR-1, CD69, SIGLEC1, FR-gamma, ADAM 8, AZU1, AREG, SDC4, DCTN2, BID, RELT, CLEC5A, APEX1, PSP-D, FGR, SELE, SELL, MESDC2, IQGAP2, CRTAM, LILRB2, TANK, CPXM1, ARSB, SLAMF1, PEBP1, STIP1, PDGF subunit B, SCARF1, DEFA1, EPHB4, ARHGAP1, CLM-1, DAB2, LYN, CASP-8, APBB1IP, ANXA11, ICAM1, PRKCQ, VCAM1, HDGF, CD2AP, TNFRSF6B, CLEC1A, TNFRSF14, TACC3, MMP-1, NRP1, ZBTB17, NADK, PLXNA4, MMP-9, NCR1, AMIGO2, FES, CD79B, TNXB, TXNDC5, TRANCE, ARG1, PCDH17, LRMP, C1QTNF1, CLM-6, CKAP4, APP, PGLYRP1, LILRA5, CLEC10A, NMNAT1, IL-6RA, ATG4A, TIMP1, COCH, DKN1A, CD1C, DECR1, DAG1, IGFBP-2, RET, GSAP, PILRB, CLEC6A, PECAM-1, PXN, ADGRG1, DPP7, TDRKH, Siglec-9, CD40-L, VEGFC, LYVE1, FADD, FCRL1, EGF, HGF, GZMH, CLEC4G, LY75, PRDX3, COL4A1, CEACAM5, SEMA7A, NUDT5, FCRL6, PAPPA, FASLG, GRN, and/or MATN3, and/or an increased concentration in the second biological sample, as compared to the first biological sample, of at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) of TMPRSS15, CCL11, FAM3B, MMP7, NCAM1, Gal-3, CCL25, THPO, hK14, KIM1, Flt3L, PLIN1, SPON2, Gal-4, FABP4, DNER, GAL, CPM, VWC2, PPY, PAM, PVR, SERPINA5, ST3GAL1, CST5, CES2, CNDP1, CX3CL1, HO-1, PRELP, ADM, VSIG2, FABP2, CEACAM5, SLITRK2, MCP-1, NTRK3, CLUL1, CXCL16, SCF, TMPRSS5, REG4, hK11, SCGB3A1, DKKL1, NEP, CPA2, Ep-CAM, THBS2, GPNMB, ITGB5, GT, APLP1, TACSTD2, NINJ1, REN, GCG, SERPINA9, KAZALD1, SERPINA12, PODXL, AMN, IGF1R, LTBP2, ANGPTL3, SCARA5, B4GAT1, ROBO2, PDGFC, CA12, DDC, EDIL3, XPNPEP2, PRTG, NQO2, AMBP, ERBB2, IL6, MCP-1, VEGFD, GDF-2, MUC-16, KLK10, FAM3C, uPA, AGR2, METRNL, RTN4R, IGF2R, NTRK2, ITGB6, SCARF2, SCGB3A2, RGMB, EZR, PROC, FURIN, PIgR, and/or SMOC2 indicates that the human subject has undergone a therapeutic response to the therapy comprising the JAK inhibitor.

In some embodiments, the method includes: measuring the concentration of at least one protein (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 proteins) selected from the group consisting of TMPRSS15, CCL11, FAM3B, MMP7, NCAM1, Gal-3, CCL25, THPO, hK14, KIM1, Flt3L, PLIN1, SPON2, Gal-4, FABP4, DNER, GAL, CPM, VWC2, PPY, PAM, PVR, SERPINA5, ST3GAL1, CST5, CES2, CNDP1, CX3CL1, HO-1, PRELP, ADM, VSIG2, FABP2, CEACAM5, SLITRK2, MCP-1, NTRK3, CLUL1, CXCL16, SCF, TMPRSS5, REG4, hK11, SCGB3A1, DKKL1, NEP, CPA2, Ep-CAM, THBS2, GPNMB, INPPL1, LAT2, CLEC7A, PPP1R9B, NEMO, SH2B3, BCR, CD5, DNAJB1, CCL17, ITGB2, BANK1, TPSAB1, YES1, LAMP3, GM-CSF-R-alpha, CNTNAP2, ZBTB16, CD163, TXLNA, MEPE, BACH1, MAX, NFKBIE, hOSCAR, LAT, PTPRJ, SIRT2, SIRPB1, AXIN1, EIF4G1, PTX3, TRIM5, IDUA, NCF2, SELP, ARHGEF12, CASP-3, CD27, MAP4K5, DAPP1, PRDX5, TLT-2, PARK7, IL2-RA, FOXO1, ST1A1, GRAP2, NBN, CD93, FCGR2A, DCTN1, IRF9, HAVCR2, CD84, STX8, LY9, ZBTB16, CD200R1, TOP2B, THY 1, PRKRA, ITGB1BP2, CD48, CD244, HCLS1, MPO, SIT1, ICAM3, SOST, DDX58, TNF-R2, TRAF2, SMAD1, LAIR-2, PIK3AP1, VSIG4, SIGLEC10, CD6, SKAP1, FCRL5, CD177, KLRD1, ERBB2IP, MILR1, MIF, SNAP23, NUB1, TIGAR, STAMPB, DSC2, LAIR1, FKBP1B, RASSF2, FATC1, CBL, IgG Fc receptor II-b, GLO1, PVALB, SCAMP3, SLAMF8, STX16, TNF-R1, DFFA, PPP1R2, ANG-1, CCL5, MAP2K6, CRKL, CD38, CXCL5, PILRA, IRAK1, CA13, STX6, PRTN3, IL-5R-alpha, ESM-1, EGLN1, CLEC1B, TYMP, SNAP29, PDGF subunit A, TNFRSF11A, gal-8, GCNT1, STK4, TNC, THBS4, CLEC4D, SIGLEC6, WASF1, WAS, COMT, RETN, SH2D1A, RNASE3, PAR-1, CD69, SIGLEC1, FR-gamma, ADAM 8, AZU1, AREG, SDC4, DCTN2, BID, and RELT in the first biological sample; and measuring the concentration of at least one protein (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 proteins) selected from the group consisting of TMPRSS15, CCL11, FAM3B, MMP7, NCAM1, Gal-3, CCL25, THPO, hK14, KIM1, Flt3L, PLIN1, SPON2, Gal-4, FABP4, DNER, GAL, CPM, VWC2, PPY, PAM, PVR, SERPINA5, ST3GAL1, CST5, CES2, CNDP1, CX3CL1, HO-1, PRELP, ADM, VSIG2, FABP2, CEACAM5, SLITRK2, MCP-1, NTRK3, CLUL1, CXCL16, SCF, TMPRSS5, REG4, hK11, SCGB3A1, DKKL1, NEP, CPA2, Ep-CAM, THBS2, GPNMB, INPPL1, LAT2, CLEC7A, PPP1R9B, NEMO, SH2B3, BCR, CD5, DNAJB1, CCL17, ITGB2, BANK1, TPSAB1, YES1, LAMP3, GM-CSF-R-alpha, CNTNAP2, ZBTB16, CD163, TXLNA, MEPE, BACH1, MAX, NFKBIE, hOSCAR, LAT, PTPRJ, SIRT2, SIRPB1, AXIN1, EIF4G1, PTX3, TRIM5, IDUA, NCF2, SELP, ARHGEF12, CASP-3, CD27, MAP4K5, DAPP1, PRDX5, TLT-2, PARK7, IL2-RA, FOXO1, ST1A1, GRAP2, NBN, CD93, FCGR2A, DCTN1, IRF9, HAVCR2, CD84, STX8, LY9, ZBTB16, CD200R1, TOP2B, THY 1, PRKRA, ITGB1BP2, CD48, CD244, HCLS1, MPO, SIT1, ICAM3, SOST, DDX58, TNF-R2, TRAF2, SMAD1, LAIR-2, PIK3AP1, VSIG4, SIGLEC10, CD6, SKAP1, FCRL5, CD177, KLRD1, ERBB2IP, MILR1, MIF, SNAP23, NUB1, TIGAR, STAMPB, DSC2, LAIR1, FKBP1B, RASSF2, FATC1, CBL, IgG Fc receptor II-b, GLO1, PVALB, SCAMP3, SLAMF8, STX16, TNF-R1, DFFA, PPP1R2, ANG-1, CCL5, MAP2K6, CRKL, CD38, CXCL5, PILRA, IRAK1, CA13, STX6, PRTN3, IL-5R-alpha, ESM-1, EGLN1, CLEC1B, TYMP, SNAP29, PDGF subunit A, TNFRSF11A, gal-8, GCNT1, STK4, TNC, THBS4, CLEC4D, SIGLEC6, WASF1, WAS, COMT, RETN, SH2D1A, RNASE3, PAR-1, CD69, SIGLEC1, FR-gamma, ADAM 8, AZU1, AREG, SDC4, DCTN2, BID, and RELT in the second biological sample, wherein a reduced concentration in the second biological sample, as compared to the first biological sample, of at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) of INPPL1, LAT2, CLEC7A, PPP1R9B, NEMO, SH2B3, BCR, CD5, DNAJB1, CCL17, ITGB2, BANK1, TPSAB1, YES1, LAMP3, GM-CSF-R-alpha, CNTNAP2, ZBTB16, CD163, TXLNA, MEPE, BACH1, MAX, NFKBIE, hOSCAR, LAT, PTPRJ, SIRT2, SIRPB1, AXIN1, EIF4G1, PTX3, TRIM5, IDUA, NCF2, SELP, ARHGEF12, CASP-3, CD27, MAP4K5, DAPP1, PRDX5, TLT-2, PARK7, IL2-RA, FOXO1, ST1A1, GRAP2, NBN, CD93, FCGR2A, DCTN1, IRF9, HAVCR2, CD84, STX8, LY9, ZBTB16, CD200R1, TOP2B, THY 1, PRKRA, ITGB1BP2, CD48, CD244, HCLS1, MPO, SIT1, ICAM3, SOST, DDX58, TNF-R2, TRAF2, SMAD1, LAIR-2, PIK3AP1, VSIG4, SIGLEC10, CD6, SKAP1, FCRL5, CD177, KLRD1, ERBB2IP, MILR1, MIF, SNAP23, NUB1, TIGAR, STAMPB, DSC2, LAIR1, FKBP1B, RASSF2, FATC1, CBL, IgG Fc receptor II-b, GLO1, PVALB, SCAMP3, SLAMF8, STX16, TNF-R1, DFFA, PPP1R2, ANG-1, CCL5, MAP2K6, CRKL, CD38, CXCL5, PILRA, IRAK1, CA13, STX6, PRTN3, IL-5R-alpha, ESM-1, EGLN1, CLEC1B, TYMP, SNAP29, PDGF subunit A, TNFRSF11A, gal-8, GCNT1, STK4, TNC, THBS4, CLEC4D, SIGLEC6, WASF1, WAS, COMT, RETN, SH2D1A, RNASE3, PAR-1, CD69, SIGLEC1, FR-gamma, ADAM 8, AZU1, AREG, SDC4, DCTN2, BID, and RELT, and/or an increased concentration in the second biological sample, as compared to the first biological sample, of at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) of TMPRSS15, CCL11, FAM3B, MMP7, NCAM1, Gal-3, CCL25, THPO, hK14, KIM1, Flt3L, PLIN1, SPON2, Gal-4, FABP4, DNER, GAL, CPM, VWC2, PPY, PAM, PVR, SERPINA5, ST3GAL1, CST5, CES2, CNDP1, CX3CL1, HO-1, PRELP, ADM, VSIG2, FABP2, CEACAM5, SLITRK2, MCP-1, NTRK3, CLUL1, CXCL16, SCF, TMPRSS5, REG4, hK11, SCGB3A1, DKKL1, NEP, CPA2, Ep-CAM, THBS2, and GPNMB indicates that the human subject has undergone a therapeutic response to the therapy comprising the JAK inhibitor.

In some embodiments, the method includes: measuring the concentration of at least one protein (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 proteins) selected from the group consisting of TMPRSS15, CCL11, FAM3B, MMP7, NCAM1, Gal-3, CCL25, THPO, hK14, KIM1, INPPL1, LAT2, CLEC7A, PPP1R9B, NEMO, SH2B3, BCR, CD5, DNAJB1, CCL17, ITGB2, BANK1, TPSAB1, YES1, LAMP3, GM-CSF-R-alpha, CNTNAP2, ZBTB16, CD163, TXLNA, MEPE, BACH1, MAX, NFKBIE, hOSCAR, LAT, PTPRJ, SIRT2, SIRPB1, AXIN1, EIF4G1, PTX3, TRIM5, IDUA, NCF2, SELP, ARHGEF12, CASP-3, CD27, MAP4K5, DAPP1, PRDX5, TLT-2, PARK7, IL2-RA, FOXO1, ST1A1, GRAP2, NBN, CD93, FCGR2A, DCTN1, IRF9, and HAVCR2 in the first biological sample; and measuring the concentration of at least one protein (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 proteins) selected from the group consisting of TMPRSS15, CCL11, FAM3B, MMP7, NCAM1, Gal-3, CCL25, THPO, hK14, KIM1, INPPL1, LAT2, CLEC7A, PPP1R9B, NEMO, SH2B3, BCR, CD5, DNAJB1, CCL17, ITGB2, BANK1, TPSAB1, YES1, LAMP3, GM-CSF-R-alpha, CNTNAP2, ZBTB16, CD163, TXLNA, MEPE, BACH1, MAX, NFKBIE, hOSCAR, LAT, PTPRJ, SIRT2, SIRPB1, AXIN1, EIF4G1, PTX3, TRIM5, IDUA, NCF2, SELP, ARHGEF12, CASP-3, CD27, MAP4K5, DAPP1, PRDX5, TLT-2, PARK7, IL2-RA, FOXO1, ST1A1, GRAP2, NBN, CD93, FCGR2A, DCTN1, IRF9, and HAVCR2 in the second biological sample, wherein a reduced concentration in the second biological sample, as compared to the first biological sample, of at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) of INPPL1, LAT2, CLEC7A, PPP1R9B, NEMO, SH2B3, BCR, CD5, DNAJB1, CCL17, ITGB2, BANK1, TPSAB1, YES1, LAMP3, GM-CSF-R-alpha, CNTNAP2, ZBTB16, CD163, TXLNA, MEPE, BACH1, MAX, NFKBIE, hOSCAR, LAT, PTPRJ, SIRT2, SIRPB1, AXIN1, EIF4G1, PTX3, TRIM5, IDUA, NCF2, SELP, ARHGEF12, CASP-3, CD27, MAP4K5, DAPP1, PRDX5, TLT-2, PARK7, IL2-RA, FOXO1, ST1A1, GRAP2, NBN, CD93, FCGR2A, DCTN1, IRF9, and HAVCR2, and/or an increased concentration in the second biological sample, as compared to the first biological sample, of at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of TMPRSS15, CCL11, FAM3B, MMP7, NCAM1, Gal-3, CCL25, THPO, hK14, and KIM1 indicates that the human subject has undergone a therapeutic response to the therapy comprising the JAK inhibitor.

In some embodiments, the method includes: measuring the concentration of at least one protein (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 proteins) selected from the group consisting of TMPRSS15, CCL11, FAM3B, MMP7, NCAM1, Gal-3, CCL25, INPPL1, LAT2, CLEC7A, PPP1R9B, NEMO, SH2B3, BCR, CD5, DNAJB1, CCL17, ITGB2, BANK1, TPSAB1, YES1, LAMP3, GM-CSF-R-alpha, and CNTNAP2 in the first biological sample; and measuring the concentration of at least one protein (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 proteins) selected from the group consisting of TMPRSS15, CCL11, FAM3B, MMP7, NCAM1, Gal-3, CCL25, INPPL1, LAT2, CLEC7A, PPP1R9B, NEMO, SH2B3, BCR, CD5, DNAJB1, CCL17, ITGB2, BANK1, TPSAB1, YES1, LAMP3, GM-CSF-R-alpha, and CNTNAP2 in the second biological sample, wherein a reduced concentration in the second biological sample, as compared to the first biological sample, of at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17) of INPPL1, LAT2, CLEC7A, PPP1R9B, NEMO, SH2B3, BCR, CD5, DNAJB1, CCL17, ITGB2, BANK1, TPSAB1, YES1, LAMP3, GM-CSF-R-alpha, and CNTNAP2, and/or an increased concentration in the second biological sample, as compared to the first biological sample, of at least one (e.g., at least 1, 2, 3, 4, 5, 6, or 7) of TMPRSS15, CCL11, FAM3B, MMP7, NCAM1, Gal-3, and CCL25 indicates that the human subject has undergone a therapeutic response to the therapy comprising the JAK inhibitor.

In some embodiments, the method includes: measuring the concentration of at least one protein (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 proteins) selected from the group consisting of TMPRSS15, CCL11, FAM3B, MMP7, NCAM1, INPPL1, LAT2, CLEC7A, PPP1R9B, NEMO, SH2B3, BCR, CD5, DNAJB1, CCL17, and ITGB2 in the first biological sample; and measuring the concentration of at least one protein (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 proteins) selected from the group consisting of TMPRSS15, CCL11, FAM3B, MMP7, NCAM1, INPPL1, LAT2, CLEC7A, PPP1R9B, NEMO, SH2B3, BCR, CD5, DNAJB1, CCL17, and ITGB2 in the second biological sample, wherein a reduced concentration in the second biological sample, as compared to the first biological sample, of at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) of INPPL1, LAT2, CLEC7A, PPP1R9B, NEMO, SH2B3, BCR, CD5, DNAJB1, CCL17, and ITGB2, and/or an increased concentration in the second biological sample, as compared to the first biological sample, of at least one (e.g., at least 1, 2, 3, 4, or 5) of TMPRSS15, CCL11, FAM3B, MMP7, and NCAM1 indicates that the human subject has undergone a therapeutic response to the therapy comprising the JAK inhibitor.

The disclosure also features a method of treating a human subject having, suspected of having, or at risk of developing GvHD, by administering to the human subject a therapy comprising a JAK inhibitor, wherein the human subject has been previously determined to have (i) a baseline concentration of at least one protein selected from the group consisting of MCP-3, CA5A, IL8, CXCL10, IL6, CCL19, CTSL1, ACE2, ALDH1A1, TNFRSF6B, KYNU, FOSB, ALDH3A1, and DDAH1 in a biological sample obtained from the human subject that is lower than a control, and/or (ii) a baseline concentration of at least one protein selected from the group consisting of PON3, SCF, GH, SRC, and CR2 in a biological sample obtained from the human subject that is higher than a control.

The disclosure also features a method of treating a human subject having, suspected of having, or at risk of developing GvHD by: providing a biological sample obtained from the human subject; measuring in the biological sample a reduced concentration, as compared to a control, of at least one protein selected from the group consisting of MCP-3, CA5A, IL8, CXCL10, IL6, CCL19, CTSL1, ACE2, ALDH1A1, TNFRSF6B, KYNU, FOSB, ALDH3A1, and DDAH1, and/or an increased concentration, as compared to a control, of at least one protein selected from the group consisting of PON3, SCF, GH, SRC, and CR2; and administering a therapy comprising a JAK inhibitor to the human subject.

The disclosure also features a method of predicting the response of a human subject having, suspected of having, or at risk of developing GvHD to a therapy comprising a JAK inhibitor by: providing a biological sample obtained from the subject before the therapy comprising the JAK inhibitor; and measuring the concentration of at least one protein selected from the group consisting of PON3, SCF, GH, SRC, CR2, MCP-3, CA5A, IL8, CXCL10, IL6, CCL19, CTSL1, ACE2, ALDH1A1, TNFRSF6B, KYNU, FOSB, ALDH3A1, and DDAH1 in the biological sample, wherein a reduced concentration, as compared to a control, of MCP-3, CA5A, IL8, CXCL10, IL6, CCL19, CTSL1, ACE2, ALDH1A1, TNFRSF6B, KYNU, FOSB, ALDH3A1, and/or DDAH1, and/or an increased concentration, as compared to a control, of PON3, SCF, GH, SRC, and/or CR2 is predictive that the subject will respond to the therapy comprising the JAK inhibitor.

In some embodiments of the methods described herein, the control is a pre-established cut-off value.

In some embodiments of the methods described herein, the control is the concentration of the protein in a sample or samples obtained from one or more subjects that have not responded to treatment with the JAK inhibitor.

The disclosure also features a method for measuring the amount of a protein in a sample by: providing a biological sample obtained from a human subject having, suspected of having, or at risk of developing GvHD; and measuring the concentration of at least one protein selected from the group consisting of PON3, SCF, GH, SRC, CR2, MCP-3, CA5A, IL8, CXCL10, IL6, CCL19, CTSL1, ACE2, ALDH1A1, TNFRSF6B, KYNU, FOSB, ALDH3A1, and DDAH1 in the biological sample.

In some embodiments of any of the methods described herein, the concentrations of no more than 20 proteins are measured.

In some embodiments of any of the methods described herein, the concentrations of no more than 10 proteins are measured.

In some embodiments of any of the methods described herein, the biological sample is blood, serum, plasma, urine, spinal fluid, saliva, lacrimal fluid, or sweat. In some embodiments, the biological sample is blood, serum, or plasma.

In some embodiments of any of the methods described herein, the concentration of the protein is measured by an immunological method. The immunological method can be, for example, an enzyme-linked immunosorbent assay, enzyme immunoassay, radioimmunoassay, chemiluminescent immunoassay, electrochemiluminescence immunoassay, latex turbidimetric immunoassay, latex photometric immunoassay, immuno-chromatographic assay, or western blotting.

In some embodiments of any of the methods described herein, the concentration of the protein is measured by mass spectrometry.

In some embodiments of any of the methods described herein, the JAK inhibitor is itacitinib.

In some embodiments of any of the methods described herein, the JAK inhibitor is 4-[3-(cyanomethyl)-3-(3′,5′-dimethyl-1H,1′H-4,4′-bipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide or a pharmaceutically acceptable salt thereof or ((2R,5S)-5-{2-[(1R)-1-hydroxyethyl]-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl}tetrahydro-2H-pyran-2-yl)acetonitrile or a pharmaceutically acceptable salt thereof.

In some embodiments of any of the methods described herein, a second therapeutic agent is administered to the human subject in combination with the JAK inhibitor. The second therapeutic agent can be, for example, a corticosteroid (e.g., methylprednisolone or prednisone), methotrexate, cyclosporine, mycophenolate mofetil, tacrolimus, sirolimus, everolimus, antithymocyte globulin, alemtuzumab, cyclophosphamide, ibrutinib, imatinib, infliximab, etanercept, tocilizumab, alemtuzumab, basiliximab, daclizumab, rituximab, denileukin diftitox, pentostatin, ciclosporin, thalidomide, halofuginone, hydroxychloroquine, or mesenchymal stem cells. The JAK inhibitor and the second therapeutic agent can be administered simultaneously or sequentially.

In some embodiments of any of the methods described herein, the GvHD is acute GvHD.

In some embodiments of any of the methods described herein, the GvHD is chronic GvHD.

The term “baseline concentration” of protein refers to the concentration of a protein in a subject prior to initiation of treatment with a JAK inhibitor.

The term “reduced concentration” means a concentration of the protein being analyzed that is lower than the concentration of that protein in a control or in a previous sample. For example, the concentration of the protein being analyzed can be at least 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 50, 75, or 100 times lower, or at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1,000%, 1,500%, 2,000%, 2,500%, 3,000%, 3,500%, 4,000%, 4,500%, or 5,000% lower, than the concentration of that protein in a control.

The term “increased concentration” means a concentration of the protein being analyzed that is higher than the concentration of that protein in a control or in a previous sample. For example, the concentration of the protein being analyzed can be at least 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 50, 75, or 100 times higher, or at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1,000%, 1,500%, 2,000%, 2,500%, 3,000%, 3,500%, 4,000%, 4,500%, or 5,000% higher, than the concentration of that protein in a control.

The term “respond to a therapy” means that the subject administered with the therapy shows a positive response to the JAK inhibitor therapy provided.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the exemplary methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present application, including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

DETAILED DESCRIPTION

This disclosure provides methods and compositions for treating a subject having, suspected of having, or at risk of developing GvHD with a JAK inhibitor. The disclosure provides predictive biomarkers (e.g., protein expression levels) to identify those subjects having, suspected of having, or at risk of developing GvHD for whom administering a therapy comprising a JAK inhibitor is likely to be effective.

Graft Versus Host Disease

GvHD occurs when donor T cells respond to genetically defined proteins (including but not limited to Human Leukocyte Antigens) on host cells. Acute GvHD is generally defined to occur prior to day 100 post-transplant, whereas chronic GvHD occurs after that time.

The clinical manifestations of acute GvHD occur in the skin, gastrointestinal tract, and liver. Skin is the most commonly affected organ in acute GvHD and is usually the first organ involved, often coinciding with engraftment of donor cells. The characteristic maculopapular rash is pruritic and can spread throughout the body. In severe cases, the skin may blister and ulcerate. Other features include dyskeratosis, exocytosis of lymphocytes, satellite lymphocytes adjacent to dyskeratotic epidermal keratinocytes, and a perivascular lymphocytic infiltration in the dermis. Gastrointestinal tract involvement of acute GvHD usually presents as diarrhea but may also include vomiting, anorexia, and/or abdominal pain. The histologic features of liver disease caused by GvHD are endothelialitis, lymphocytic infiltration of the portal areas, pericholangitis, and bile duct destruction.

Chronic GvHD is the major cause of late non-relapse death following hematopoietic cell transplant. Its presentation may be progressive (e.g., acute GvHD merging into chronic GvHD), quiescent (acute GvHD that resolves completely but is later followed by chronic GvHD), or it may occur de novo. Older recipient age and a history of acute GvHD are the greatest risk factors for chronic GvHD. Clinical signs of chronic GvHD often first appear in the buccal mucosa.

Methods of Predicting Responsiveness to a Therapy Comprising a JAK Inhibitor Several proteins have been identified in the Examples whose expression levels are useful in predicting responsiveness (e.g., improvement in disease scores and/or disease resolution) of a subject having GvHD to a therapy comprising a JAK inhibitor. These proteins are listed in Tables 1 and 2.

TABLE 1 Biomarkers Exhibiting Reduced Expression in GvHD Subjects that Respond to Treatment with a JAK inhibitor as Compared to Control Subjects that do not Respond Protein   IL8 HAOX1 ENPP7 ACE2 SULT2A1 MCP-3 CES1 MFGE8 PLXNB1 TNFRSF10A CCL15 TNFRSF10A SEMA4C PREB NFATC3 CCL19 DLL1 ENTPD2 IL-4RA EPHA2 FOSB CXCL10 VAMP5 ALDH3A1 MVK IL12RB1 CALCA AHCY PRSS2 LILRB4 DDAH1 IL-1ra NECTIN2 PDCD1 CD74 PD-L1 REG3A CA5A N2DL-2 CDCP1 U-PAR SIGLEC7 ANGPTL4 ALDH1A1 SPINK1 HTRA2 PRDX6 IL-1RT2 IGFBP-1 HNMT TRAIL-R2 CXADR CTSL1 IFN-gamma-R1 IL-18R1 KRT19 KYNU TGM2

TABLE 2 Biomarkers Exhibiting Increased Expression in GvHD Subjects that Respond to Treatment with a JAK inhibitor as Compared to Control Subjects that do not Respond Protein   PON3 CNTN1 IGFBP3 LEP Notch 3 TN-R HSD11B1 FAM19A5 NCAN F11 GDF-8 CCL28 GALNT10 BCAN TIMP4 CRISP2 CD207 WNT9A MBL2 EN-RAGE TWEAK CR2 MFAP5 KIT GH PFKM CDSN CRH GCP5 KLK6 DRAXIN

A reduced protein concentration compared to a control of one or more (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) proteins listed in Table 1 is indicative/predictive that a subject that has, is suspected of having, or is at risk of developing GvHD will respond to a therapy comprising a JAK inhibitor. For example, low concentrations (compared to a control) of CXCL10 protein in a biological sample obtained from a subject prior to treatment with the therapy comprising a JAK inhibitor are predictive that the subject will respond to the therapy comprising a JAK inhibitor.

An increased protein concentration compared to a control of one or more (e.g., at least 1, 2, 3, 4, or 5) proteins listed in Table 2 is indicative/predictive that a subject that has, is suspected of having, or is at risk of developing GvHD will respond to a therapy comprising a JAK inhibitor. For example, increased concentrations (compared to a control) of PON3 protein in a biological sample obtained from a subject prior to treatment with the therapy comprising a JAK inhibitor are predictive that the subject will respond to the therapy comprising a JAK inhibitor.

A reduced protein concentration compared to a control of one or more (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) proteins listed in Table 1 combined with an increased protein concentration compared to a control of one or more (e.g., at least 1, 2, 3, 4, or 5) proteins listed in Table 2 is indicative/predictive that a subject that has, is suspected of having, or is at risk of developing GvHD will respond to a therapy comprising a JAK inhibitor. For example, low concentrations (compared to a control) of CXCL10 protein and increased concentrations (compared to a control) of PON3 protein in a biological sample obtained from a subject prior to treatment with the therapy comprising a JAK inhibitor are predictive that the subject will respond to the therapy comprising a JAK inhibitor. In another example, low concentrations (compared to a control) of MCP-3, CA5A, IL8, CXCL10, IL6, CCL19, CTSL1, ACE2, ALDH1A1, TNFRSF6B, KYNU, FOSB, ALDH3A1, and DDAH1 proteins and increased concentrations (compared to a control) of PON3, SCF, GH, SRC, and CR2 proteins in a biological sample obtained from a subject prior to treatment with the therapy comprising a JAK inhibitor are predictive that the subject will respond to the therapy comprising a JAK inhibitor.

In some embodiments, the GvHD is acute GvHD. In other embodiments, the GvHD is chronic GvHD.

Controls

As described above, the methods of the present invention can involve, measuring the concentration of one or more proteins (e.g., one or more proteins depicted in Table 1 and/or Table 2) in a biological sample from a subject having, suspected of having or at risk of developing GvHD, wherein the concentration of one or more proteins, compared to a control, predicts the response of a subject to treatment comprising a JAK inhibitor. In certain embodiments, when the concentration of a protein in Table 1 in a biological sample from a subject having, suspected of having or at risk of developing GvHD is lower than the control, the subject is identified as likely to respond to a therapy comprising a JAK inhibitor. In other embodiments, when the concentration of a protein in Table 2 in a biological sample from a subject having, suspected of having or at risk of developing GvHD is higher than the control, the subject is identified as likely to respond to a therapy comprising a JAK inhibitor. In this context, the term “control” includes a sample (from the same tissue type) obtained from a subject who is known to not respond to a therapy comprising a JAK inhibitor. The term “control” also includes a sample (from the same tissue type) obtained in the past from a subject who is known to not respond to a therapy comprising a JAK inhibitor and used as a reference for future comparisons to test samples taken from subjects for which therapeutic responsiveness is to be predicted. The “control” expression level/concentration for a particular protein in a particular cell type or tissue may be pre-established by an analysis of protein expression in one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, or 40 or more) subjects, of the same species, that have not responded to treatment with a JAK inhibitor. This pre-established reference value (which may be an average or median expression level/concentration taken from multiple subjects that have not responded to the therapy) may then be used for the “control” concentration/expression level of the protein in the comparison with the test sample. In such a comparison, the subject is predicted to respond to a therapy comprising a JAK inhibitor if the expression level of the protein being analyzed is lower (Table 1) or higher (Table 2) than the pre-established reference.

The “control” concentration for a particular protein in a particular cell type or tissue may alternatively be pre-established by an analysis of protein expression in one or more subjects that have responded to treatment with a JAK inhibitor. This pre-established reference value (which may be an average or median expression level taken from multiple subjects that have responded to the therapy) may then be used as the “control” expression level in the comparison with the test sample. In such a comparison, the subject is predicted to respond to a therapy comprising a JAK inhibitor if the concentration of the protein being analyzed is the same as, or comparable to (e.g., at least 85% but less than 100% of), the pre-established reference.

In certain embodiments, the “control” is a pre-established cut-off value. A cut-off value is typically a concentration of a protein above or below which is considered predictive of responsiveness of a subject to a therapy of interest. Thus, in accordance with the methods and compositions described herein, a reference protein concentration (e.g., of a protein of Table 1 or Table 2) is identified as a cut-off value, above or below of which is predictive of responsiveness to a therapy comprising a JAK inhibitor. Cut-off values determined for use in the methods described herein can be compared with, e.g., published ranges of concentrations but can be individualized to the methodology used and patient population.

In some embodiments, the concentration of the protein being analyzed is reduced as compared to the concentration of that protein in a control. For example, the concentration of the protein being analyzed can be at least 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 50, 75, or 100 times lower, or at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1,000%, 1,500%, 2,000%, 2,500%, 3,000%, 3,500%, 4,000%, 4,500%, or 5,000% lower, than the concentration of that protein in a control.

In some embodiments, the concentration of the protein being analyzed is increased as compared to the concentration of that protein in a control. For example, the concentration of the protein being analyzed can be at least 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 50, 75, or 100 times higher, or at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1,000%, 1,500%, 2,000%, 2,500%, 3,000%, 3,500%, 4,000%, 4,500%, or 5,000% higher, than the concentration of that protein in a control.

Methods of Identifying Therapeutic Responsiveness to a Therapy Comprising a JAK Inhibitor

Several proteins have been identified in the Examples whose expression levels, in subjects who respond to treatment with a JAK inhibitor, change during the course of treatment and are therefore useful in identifying therapeutic responsiveness (e.g., improvement in disease scores and/or disease resolution) of a subject having GvHD to a therapy comprising a JAK inhibitor. These proteins are identified in Table 13.

A reduced protein concentration in a biological sample obtained from a subject after treatment with a JAK inhibitor, as compared to the baseline expression level in a biological sample obtained from the subject before treatment with a JAK inhibitor, of one or more (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) of INPPL1, LAT2, CLEC7A, PPP1R9B, NEMO, SH2B3, BCR, CD5, DNAJB1, CCL17, ITGB2, BANK1, TPSAB1, YES1, LAMP3, GM-CSF-R-alpha, CNTNAP2, ZBTB16, CD163, TXLNA, MEPE, BACH1, MAX, NFKBIE, hOSCAR, LAT, PTPRJ, SIRT2, SIRPB1, AXIN1, EIF4G1, PTX3, TRIM5, IDUA, NCF2, SELP, ARHGEF12, CASP-3, CD27, MAP4K5, DAPP1, PRDX5, TLT-2, PARK7, IL2-RA, FOXO1, ST1A1, GRAP2, NBN, CD93, FCGR2A, DCTN1, IRF9, HAVCR2, CD84, STX8, LY9, ZBTB16, CD200R1, TOP2B, THY 1, PRKRA, ITGB1BP2, CD48, CD244, HCLS1, MPO, SIT1, ICAM3, SOST, DDX58, TNF-R2, TRAF2, SMAD1, LAIR-2, PIK3AP1, VSIG4, SIGLEC10, CD6, SKAP1, FCRL5, CD177, KLRD1, ERBB2IP, MILR1, MIF, SNAP23, NUB1, TIGAR, STAMPB, DSC2, LAIR1, FKBP1B, RASSF2, FATC1, CBL, IgG Fc receptor II-b, GLO1, PVALB, SCAMP3, SLAMF8, STX16, TNF-R1, DFFA, PPP1R2, ANG-1, CCL5, MAP2K6, CRKL, CD38, CXCL5, PILRA, IRAK1, CA13, STX6, PRTN3, IL-5R-alpha, ESM-1, EGLN1, CLEC1B, TYMP, SNAP29, PDGF subunit A, TNFRSF11A, gal-8, GCNT1, STK4, TNC, THBS4, CLEC4D, SIGLEC6, WASF1, WAS, COMT, RETN, SH2D1A, RNASE3, PAR-1, CD69, SIGLEC1, FR-gamma, ADAM 8, AZU1, AREG, SDC4, DCTN2, BID, RELT, CLEC5A, APEX1, PSP-D, FGR, SELE, SELL, MESDC2, IQGAP2, CRTAM, LILRB2, TANK, CPXM1, ARSB, SLAMF1, PEBP1, STIP1, PDGF subunit B, SCARF1, DEFA1, EPHB4, ARHGAP1, CLM-1, DAB2, LYN, CASP-8, APBB1IP, ANXA11, ICAM1, PRKCQ, VCAM1, HDGF, CD2AP, TNFRSF6B, CLEC1A, TNFRSF14, TACC3, MMP-1, NRP1, ZBTB17, NADK, PLXNA4, MMP-9, NCR1, AMIGO2, FES, CD79B, TNXB, TXNDC5, TRANCE, ARG1, PCDH17, LRMP, C1QTNF1, CLM-6, CKAP4, APP, PGLYRP1, LILRA5, CLEC10A, NMNAT1, IL-6RA, ATG4A, TIMP1, COCH, DKN1A, CD1C, DECR1, DAG1, IGFBP-2, RET, GSAP, PILRB, CLEC6A, PECAM-1, PXN, ADGRG1, DPP7, TDRKH, Siglec-9, CD40-L, VEGFC, LYVE1, FADD, FCRL1, EGF, HGF, GZMH, CLEC4G, LY75, PRDX3, COL4A1, CEACAM8, SEMA7A, NUDT5, FCRL6, PAPPA, FASLG, GRN, and/or MATN3 is indicative that the subject has undergone a therapeutic response to the JAK inhibitor.

An increased protein concentration in a biological sample obtained from a subject after treatment with a JAK inhibitor, as compared to the baseline expression level in a biological sample obtained from the subject before treatment with a JAK inhibitor, of one or more (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) of TMPRSS15, CCL11, FAM3B, MMP7, NCAM1, Gal-3, CCL25, THPO, CCL11, hK14, KIM1, Flt3L, PLIN1, SPON2, Gal-4, FABP4, DNER, GAL, CPM, VWC2, PPY, PAM, PVR, SERPINA5, ST3GAL1, CST5, CES2, CNDP1, CX3CL1, HO-1, PRELP, ADM, VSIG2, FABP2, CEACAM5, SLITRK2, MCP-1, NTRK3, CLUL1, CXCL16, SCF, TMPRSS5, REG4, hK11, SCGB3A1, DKKL1, NEP, CPA2, Ep-CAM, THBS2, GPNMB, ITGB5, GT, APLP1, TACSTD2, NINJ1, REN, GCG, SERPINA9, KAZALD1, SERPINA12, PODXL, AMN, IGF1R, LTBP2, ANGPTL3, SCARA5, B4GAT1, ROBO2, PDGFC, CA12, DDC, EDIL3, XPNPEP2, PRTG, NQO2, AMBP, ERBB2, IL6, MCP-1, VEGFD, GDF-2, MUC-16, KLK10, FAM3C, uPA, AGR2, METRNL, RTN4R, IGF2R, NTRK2, ITGB6, SCARF2, SCGB3A2, RGMB, EZR, PROC, FURIN, PIgR, and/or SMOC2 is indicative that the subject has undergone a therapeutic response to the JAK inhibitor.

A reduced protein concentration in a biological sample obtained from a subject after treatment with a JAK inhibitor, as compared to the baseline expression level in a biological sample obtained from the subject before treatment with a JAK inhibitor, of one or more (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) of INPPL1, LAT2, CLEC5A, PPP1R9B, NEMO, SH2B3, BCR, CD5, DNAJB1, CCL17, ITGB2, BANK1, TPSAB1, YES1, LAMP3, GM-CSF-R-alpha, CNTNAP2, ZBTB16, CD163, TXLNA, MEPE, BACH1, MAX, NFKBIE, hOSCAR, LAT, PTPRJ, SIRT2, SIRPB1, AXIN1, EIF4G1, PTX3, TRIM5, IDUA, NCF2, SELP, ARHGEF12, CASP-3, CD27, MAP4K5, DAPP1, PRDX5, TLT-2, PARK7, IL2-RA, FOXO1, ST1A1, GRAP2, NBN, CD93, FCGR2A, DCTN1, IRF9, HAVCR2, CD84, STX8, LY9, ZBTB16, CD200R1, TOP2B, THY 1, PRKRA, ITGB1BP2, CD48, CD244, HCLS1, MPO, SIT1, ICAM3, SOST, DDX58, TNF-R2, TRAF2, SMAD1, LAIR-2, PIK3AP1, VSIG4, SIGLEC10, CD6, SKAP1, FCRL5, CD177, KLRD1, ERBB2IP, MILR1, MIF, SNAP23, NUB1, TIGAR, STAMPB, DSC2, LAIR1, FKBP1B, RASSF2, FATC1, CBL, IgG Fc receptor II-b, GLO1, PVALB, SCAMP3, SLAMF8, STX16, TNF-R1, DFFA, PPP1R2, ANG-1, CCL5, MAP2K6, CRKL, CD38, CXCL5, PILRA, IRAK1, CA13, STX6, PRTN3, IL-5R-alpha, ESM-1, EGLN1, CLEC1B, TYMP, SNAP29, PDGF subunit A, TNFRSF11A, gal-8, GCNT1, STK4, TNC, THBS4, CLEC4D, SIGLEC6, WASF1, WAS, COMT, RETN, SH2D1A, RNASE3, PAR-1, CD69, SIGLEC1, FR-gamma, ADAM 8, AZU1, AREG, SDC4, DCTN2, BID, RELT, CLEC5A, APEX1, PSP-D, FGR, SELE, SELL, MESDC2, IQGAP2, CRTAM, LILRB2, TANK, CPXM1, ARSB, SLAMF1, PEBP1, STIP1, PDGF subunit B, SCARF1, DEFA1, EPHB4, ARHGAP1, CLM-1, DAB2, LYN, CASP-8, APBB1IP, ANXA11, ICAM1, PRKCQ, VCAM1, HDGF, CD2AP, TNFRSF6B, CLEC1A, TNFRSF14, TACC3, MMP-1, NRP1, ZBTB17, NADK, PLXNA4, MMP-9, NCR1, AMIGO2, FES, CD79B, TNXB, TXNDC5, TRANCE, ARG1, PCDH17, LRMP, C1QTNF1, CLM-6, CKAP4, APP, PGLYRP1, LILRA5, CLEC10A, NMNAT1, IL-6RA, ATG4A, TIMP1, COCH, DKN1A, CD1C, DECR1, DAG1, IGFBP-2, RET, GSAP, PILRB, CLEC6A, PECAM-1, PXN, ADGRG1, DPP7, TDRKH, Siglec-9, CD40-L, VEGFC, LYVE1, FADD, FCRL1, EGF, HGF, GZMH, CLEC4G, LY75, PRDX3, COL4A1, CEACAM8, SEMA7A, NUDT5, FCRL6, PAPPA, FASLG, GRN, and/or MATN3 combined with an increased protein concentration in a biological sample obtained from the subject after treatment with a JAK inhibitor, as compared to the baseline expression level in a biological sample obtained from the subject before treatment with a JAK inhibitor, of one or more (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) of TMPRSS15, CCL11, FAM3B, MMP7, NCAM1, Gal-3, CCL25, THPO, CCL11, hK14, KIM1, Flt3L, PLIN1, SPON2, Gal-4, FABP4, DNER, GAL, CPM, VWC2, PPY, PAM, PVR, SERPINA5, ST3GAL1, CST5, CES2, CNDP1, CX3CL1, HO-1, PRELP, ADM, VSIG2, FABP2, CEACAM5, SLITRK2, MCP-1, NTRK3, CLUL1, CXCL16, SCF, TMPRSS5, REG4, hK11, SCGB3A1, DKKL1, NEP, CPA2, Ep-CAM, THBS2, GPNMB, ITGB5, GT, APLP1, TACSTD2, NINE, REN, GCG, SERPINA9, KAZALD1, SERPINA12, PODXL, AMN, IGF1R, LTBP2, ANGPTL3, SCARA5, B4GAT1, ROBO2, PDGFC, CA12, DDC, EDIL3, XPNPEP2, PRTG, NQO2, AMBP, ERBB2, IL6, MCP-1, VEGFD, GDF-2, MUC-16, KLK10, FAM3C, uPA, AGR2, METRNL, RTN4R, IGF2R, NTRK2, ITGB6, SCARF2, SCGB3A2, RGMB, EZR, PROC, FURIN, PIgR, and/or SMOC2 is indicative that the subject has undergone a therapeutic response to the JAK inhibitor.

In some embodiments, the GvHD is acute GvHD. In other embodiments, the GvHD is chronic GvHD.

Biological Samples

Suitable biological samples for the methods described herein include any biological fluid, cell, tissue, or fraction thereof, which includes proteins of interest. A biological sample can be, for example, a specimen obtained from a human subject or can be derived from such a subject. For example, a biological sample can be a biological fluid such as blood, serum, plasma, urine, spinal fluid, saliva, lacrimal fluid, or sweat, or such a sample absorbed onto a substrate (e.g., glass, polymer, or paper).

A biological sample can be obtained from a subject having, suspected of having, or at risk of developing, GvHD. In certain embodiments, the subject has acute GvHD. In some embodiments, the subject has chronic GvHD.

Methods for obtaining and/or storing samples that preserve the activity or integrity of molecules (e.g., proteins) in the sample are well known to those skilled in the art. For example, a biological sample can be further contacted with one or more additional agents such as buffers and/or inhibitors, including one or more of nuclease, protease, and phosphatase inhibitors, which preserve or minimize changes in the molecules in the sample.

Determining Expression Levels/Concentrations of Biomarkers

The presence or expression level (amount) of a gene can be determined by detecting and/or measuring the level of protein expression of the gene.

In one embodiment, the expression of a gene can be determined by detecting and/or measuring expression or concentration of a protein encoded by the gene. Methods of determining protein expression/concentration are well known in the art. A generally used method involves the use of antibodies specific for the target protein of interest. For example, methods of determining protein expression include, but are not limited to, western blot or dot blot analysis, immunohistochemistry (e.g., quantitative immunohistochemistry), immunocytochemistry, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunosorbent spot (ELISPOT; Coligan, J. E., et al., eds. (1995) Current Protocols in Immunology. Wiley, New York), radioimmunoassay, chemiluminescent immunoassay, electrochemiluminescence immunoassay, latex turbidimetric immunoassay, latex photometric immunoassay, immuno-chromatographic assay, and antibody array analysis (see, e.g., U.S. Publication Nos. 20030013208 and 2004171068, the disclosures of each of which are incorporated herein by reference in their entirety).

In one example, the presence or amount of protein expression of a gene (e.g., a gene depicted in Table 1, Table 2, or Table 13) can be determined using a western blotting technique. For example, a lysate can be prepared from a biological sample, or the biological sample itself, can be contacted with Laemmli buffer and subjected to sodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE-resolved proteins, separated by size, can then be transferred to a filter membrane (e.g., nitrocellulose) and subjected to immunoblotting techniques using a detectably-labeled antibody specific to the protein of interest. The presence or amount of bound detectably-labeled antibody indicates the presence or amount of protein in the biological sample.

In another example, an immunoassay can be used for detecting and/or measuring the protein expression of a gene (e.g., a gene depicted in Table 1, Table 2, or Table 13). As above, for the purposes of detection, an immunoassay can be performed with an antibody that bears a detection moiety (e.g., a fluorescent agent or enzyme). Proteins from a biological sample can be conjugated directly to a solid-phase matrix (e.g., a multi-well assay plate, nitrocellulose, agarose, sepharose, encoded particles, or magnetic beads) or it can be conjugated to a first member of a specific binding pair (e.g., biotin or streptavidin) that attaches to a solid-phase matrix upon binding to a second member of the specific binding pair (e.g., streptavidin or biotin). Such attachment to a solid-phase matrix allows the proteins to be purified away from other interfering or irrelevant components of the biological sample prior to contact with the detection antibody and also allows for subsequent washing of unbound antibody. Here as above, the presence or amount of bound detectably-labeled antibody indicates the presence or amount of protein in the biological sample.

There is no particular restriction as to the form of the antibody and the present disclosure includes polyclonal antibodies, as well as monoclonal antibodies. The antiserum obtained by immunizing animals, such as rabbits with a protein or fragment thereof (i.e., a protein or an immunological fragment thereof from Table 1, Table 2, or Table 13), as well polyclonal and monoclonal antibodies of all classes, human antibodies, and humanized antibodies produced by genetic recombination, are also included. Antibodies or antibody fragments specific for a protein encoded by one or more biomarkers can also be generated by in vitro methods such as phage display. Moreover, the antibody may be an antibody fragment or modified-antibody, so long as it binds to a protein encoded by a biomarker of the invention. For instance, Fab, F (ab′) 2, Fv, or single chain Fv (scFv) in which the H chain Fv and the L chain Fv are suitably linked by a linker (Huston et al., Proc. Natl. Acad. Sci. USA, 85:5879-5883, (1988)) can be given as antibody fragments.

The antibodies may be conjugated to various molecules, such as fluorescent substances, radioactive substances, and luminescent substances. Methods to attach such moieties to an antibody are already established and conventional in the field (see, e.g., U.S. Pat. Nos. 5,057,313 and 5,156,840).

Examples of methods that assay the antigen-binding activity of the antibodies include, for example, measurement of absorbance, enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), and/or immunofluorescence. For example, when using ELISA, a protein encoded by a biomarker of the invention is added to a plate coated with the antibodies of the present disclosure, and then, the antibody sample, for example, culture supernatants of antibody-producing cells, or purified antibodies are added. Then, secondary antibody recognizing the primary antibody, which is labeled by alkaline phosphatase and such enzymes, is added, the plate is incubated and washed, and the absorbance is measured to evaluate the antigen-binding activity after adding an enzyme substrate such as p-nitrophenyl phosphate. As the protein, a protein fragment, for example, a fragment comprising a C-terminus, or a fragment comprising an N-terminus may be used. To evaluate the activity of the antibody of the invention, BIAcore (GE Healthcare) may be used.

By using these methods, the antibody and a sample presumed to contain a protein of interest are contacted, and the protein encoded by a biomarker of the invention is detected or assayed by detecting or assaying the immune complex formed between the above-mentioned antibody and the protein.

Mass spectrometry based quantitation assay methods, for example, but not limited to, multiple reaction monitoring (MRM)-based approaches in combination with stable-isotope labeled internal standards, are an alternative to immunoassays for quantitative measurement of proteins. These approaches do not require the use of antibodies (see, for example, Addona et al., Nat. Biotechnol., 27:633-641, 2009; Kuzyk et al., Mol. Cell Proteomics, 8:1860-1877, 2009; Paulovich et al., Proteomics Clin. Appl., 2:1386-1402, 2008). In addition, MRM offers superior multiplexing capabilities, allowing for the simultaneous quantification of numerous proteins in parallel. The basic theory of these methods has been well-established and widely utilized for drug metabolism and pharmacokinetics analysis of small molecules.

In some embodiments, the concentration of two proteins, three proteins, four proteins, five proteins, six proteins, seven proteins, eight proteins, nine proteins, 10 proteins, 11 proteins, 12 proteins, 13 proteins, or 14 proteins, or at least two proteins, at least three proteins, at least four proteins, at least five proteins, at least six proteins, at least seven proteins, at least eight proteins, at least nine proteins, at least 10 proteins, at least 11 proteins, at least 12 proteins, at least 13 proteins, or at least 14 proteins from Table 1 can be assessed and/or measured.

In some embodiments, the concentration of two proteins, three proteins, four proteins, or five proteins, or at least two proteins, at least three proteins, at least four proteins, or at least five proteins from Table 2 can be assessed and/or measured.

In some embodiments, the concentration of two proteins, three proteins, four proteins, five proteins, six proteins, seven proteins, eight proteins, nine proteins, 10 proteins, 11 proteins, 12 proteins, 13 proteins, 14 proteins, 15 proteins, 16 proteins, 17 proteins, 18 proteins, 19 proteins, or 20 proteins, or at least two proteins, at least three proteins, at least four proteins, at least five proteins, at least six proteins, at least seven proteins, at least eight proteins, at least nine proteins, at least 10 proteins, at least 11 proteins, at least 12 proteins, at least 13 proteins, at least 14 proteins, at least 15 proteins, at least 16 proteins, at least 17 proteins, at least 18 proteins, at least 19 proteins, or at least 20 proteins from Table 13 can be assessed and/or measured.

In some embodiments of the methods described herein, the method includes measuring a concentration of MCP-3 that is below 15 pg/ml, below 10 pg/ml, below 9 pg/ml, below 8 pg/ml, below 7 pg/ml, below 6 pg/ml, below 5 pg/ml, below 4 pg/ml, or below 3 pg/ml.

In some embodiments of the methods described herein, the method includes measuring a concentration of Reg3A that is below 45,000 pg/ml, below 40,000 pg/ml, below 35,000 pg/ml, below 30,000 pg/ml, below 25,000 pg/ml, below 20,000 pg/ml, below 15,000 pg/ml, or below 10,000 pg/ml.

In some embodiments of the methods described herein, the method includes measuring a concentration of TNFRSF6B that is below 400 pg/ml, below 350 pg/ml, below 300 pg/ml, below 250 pg/ml, or below 200 pg/ml.

In some embodiments of the methods described herein, the method includes measuring a concentration of SCF that is above 350 pg/ml, above 400 pg/ml, above 450 pg/ml, above 500 pg/ml, above 600 pg/ml, or above 650 pg/ml.

In some embodiments of the methods described herein, the method includes measuring a concentration of CXCL10 that is below 900 pg/ml, below 800 pg/ml, below 700 pg/ml, below 600 pg/ml, below 500 pg/ml, or below 400 pg/ml.

In some embodiments of the methods described herein, the method includes measuring a concentration of IL-8 that is below 40 pg/ml, below 35 pg/ml, below 30 pg/ml, below 25 pg/ml, below 20 pg/ml, below 15 pg/ml, or below 10 pg/ml.

In some embodiments of the methods described herein, the method includes measuring a concentration of ST2 that is below 140,000 pg/ml, below 130,000 pg/ml, below 120,000 pg/ml, below 110,000 pg/ml, below 100,000 pg/ml, below 90,000 pg/ml, below 80,000 pg/ml, or below 70,000 pg/ml.

In some embodiments of the methods described herein, the method includes measuring a concentration of CALCA that is below 3,000 pg/ml, below 2,900 pg/ml, below 2,800 pg/ml, below 2,700 pg/ml, below 2,600 pg/ml, below 2,500 pg/ml, below 2,400 pg/ml, below 2,300 pg/ml, below 2,200 pg/ml, below 2,100 pg/ml, or below 2,000 pg/ml.

In some embodiments of the methods described herein, the method includes measuring a concentration of TNF-R1 that is below 12,000 pg/ml, below 11,500 pg/ml, below 11,000 pg/ml, below 10,500 pg/ml, below 10,000 pg/ml, or below 9,500 pg/ml.

In some embodiments of the methods described herein, the method includes measuring a concentration of IL-6 that is below 3.5 pg/ml, below 3 pg/ml, below 2.5 pg/ml, below 2 pg/ml, or below 1.5 pg/ml.

In some embodiments of the methods described herein, the method includes measuring a concentration of CCL19 that is below 1,000 pg/ml, below 900 pg/ml, below 800 pg/ml, below 700 pg/ml, below 600 pg/ml, or below 500 pg/ml.

In some embodiments of the methods described herein, the method includes measuring a concentration of PON3 that is above 150,000 pg/ml, above 200,000 pg/ml, above 250,000 pg/ml, above 300,000 pg/ml, above 350,000 pg/ml, or above 400,000 pg/ml.

JAK Inhibitors

In some embodiments, the JAK inhibitor is a compound that inhibits JAK1, JAK2, JAK3, and/or TYK2. In some embodiments, the JAK inhibitor is selective for JAK1 and JAK2 over JAK3 and TYK2. In some embodiments, the JAK inhibitor is selective for JAK1 over JAK2, JAK3, and TYK2. For example, some of the compounds described herein, or a pharmaceutically acceptable salt thereof, preferentially inhibit JAK1 over one or more of JAK2, JAK3, and TYK2. In some embodiments, the compounds or salts inhibit JAK1 preferentially over JAK2 (e.g., have a JAK2/JAK1 IC₅₀ ratio>1). In some embodiments, the compounds or salts are about 10-fold more selective for JAK1 over JAK2. In some embodiments, the compounds or salts are about 3-fold, about 5-fold, about 10-fold, about 15-fold, or about 20-fold more selective for JAK1 over JAK2 as calculated by measuring IC₅₀ at 1 mM ATP.

In some embodiments, the JAK inhibitor is 3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile.

In some embodiments, the JAK inhibitor is (3R)-3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile (ruxolitinib; also known as INCB018424).

3-Cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile and ruxolitinib can be made by the procedure described in U.S. Pat. No. 7,598,257 (Example 67), filed Dec. 12, 2006, which is incorporated herein by reference in its entirety.

In some embodiments, the JAK inhibitor is (3R)-3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile phosphoric acid salt.

In some embodiments, the JAK inhibitor is baricitinib, tofacitinib, oclacitinib, filgotinib, gandotinib, lestaurtinib, momelotinib, bacritinib, PF-04965842, upadacitinib, peficitinib, fedratinib, cucurbitacin I, ATI-501 (Aclaris), ATI-502 (Aclaris), JTE052 (Leo Pharma and Japan Tobacco), or CHZ868.

In some embodiments, the JAK inhibitor can be an isotopically-labeled compound, or a pharmaceutically acceptable salt thereof. An “isotopically” or “radio-labeled” compound is a compound of the disclosure where one or more atoms are replaced or substituted by an atom having an atomic mass or mass number different from the atomic mass or mass number typically found in nature (i.e., naturally occurring). Suitable radionuclides that may be incorporated in compounds of the present disclosure include but are not limited to ²H (also written as D for deuterium), ³H (also written as T for tritium), ¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, ¹⁸F, ³⁵S, ³⁶Cl, ⁸²Br, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ¹²³I, ¹²⁴I and ¹³¹I. For example, one or more hydrogen atoms in a compound of the present disclosure can be replaced by deuterium atoms (e.g., one or more hydrogen atoms of a C₁₋₆ alkyl group of Formula (I) can be optionally substituted with deuterium atoms, such as —CD₃ being substituted for —CH₃).

One or more constituent atoms of the compounds described herein can be replaced or substituted with isotopes of the atoms in natural or non-natural abundance. In some embodiments, the compound includes at least one deuterium atom. In some embodiments, the compound includes two or more deuterium atoms. In some embodiments, the compound includes 1-2, 1-3, 1-4, 1-5, or 1-6 deuterium atoms. In some embodiments, all of the hydrogen atoms in a compound can be replaced or substituted by deuterium atoms.

Synthetic methods for including isotopes into organic compounds are known in the art (Deuterium Labeling in Organic Chemistry by Alan F. Thomas (New York, N.Y., Appleton-Century-Crofts, 1971; The Renaissance of H/D Exchange by Jens Atzrodt, Volker Derdau, Thorsten Fey and Jochen Zimmermann, Angew. Chem. Int. Ed. 2007, 7744-7765; The Organic Chemistry of Isotopic Labelling by James R. Hanson, Royal Society of Chemistry, 2011). Isotopically labeled compounds can be used in various studies such as NMR spectroscopy, metabolism experiments, and/or assays.

Substitution with heavier isotopes, such as deuterium, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances. (see e.g., A. Kerekes et. al. J. Med. Chem. 2011, 54, 201-210; R. Xu et. al. J. Label Compd. Radiopharm. 2015, 58, 308-312). In particular, substitution at one or more metabolism sites may afford one or more of the therapeutic advantages.

Accordingly, in some embodiments, the JAK inhibitor is a compound, wherein one or more hydrogen atoms in the compound are replaced by deuterium atoms, or a pharmaceutically acceptable salt thereof.

In some embodiments, the JAK inhibitor is ruxolitinib, wherein one or more hydrogen atoms are replaced by deuterium atoms, or a pharmaceutically acceptable salt thereof. In some embodiments, the JAK inhibitor is any of the compounds in U.S. Pat. No. 9,249,149 (which is incorporated herein by reference in its entirety), or a pharmaceutically acceptable salt thereof. In some embodiments, the JAK inhibitor is CTP-543, or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is a compound of Formula I:

or a pharmaceutically acceptable salt thereof, wherein:

R¹ is selected from H and D;

each R² is independently selected from H and D, provided that each R² attached to a common carbon is the same;

each R³ is independently selected from H and D, provided that each R³ attached to a common carbon is the same;

R⁴ is selected from H and D;

each R⁵ is the same and is selected from H and D; and

R⁶, R⁷, and R⁸ are each independently selected from H and D; provided that when R¹ is H, each R² and each R³ are H, R⁴ is H, and each of R⁶, R⁷, and R⁸ is H, then each R⁵ is D.

In some embodiments, the JAK inhibitor is a compound of Formula I selected from the following compounds 100-130 in the table below (wherein R⁶, R⁷, and R⁸ are each H), or a pharmaceutically acceptable salt thereof. In some embodiments, the JAK inhibitor is a compound of Formula I selected from the following compounds 200-231 in the table below (wherein R⁶, R⁷, and R⁸ are each D), or a pharmaceutically acceptable salt thereof.

Compound R¹ Each R² Each R³ R⁴ Each R⁵ 100 H H H D H 101 H H H H D 102 H H H D D 103 H H D H H 104 H H D D H 105 H H D H D 106 H H D D D 107 H D H H H 108 H D H D H 109 H D H H D 110 H D H D D 111 H D D H H 112 H D D D H 113 H D D H D 114 H D D D D 115 D H H H H 116 D H H D H 117 D H H H D 118 D H H D D 119 D H D H H 120 D H D D H 121 D H D H D 122 D H D D D 123 D D H H H 124 D D H D H 125 D D H H D 126 D D H D D 127 D D D H H 128 D D D D H 129 D D D H D 130 D D D D D 200 H H H D H 201 H H H H D 202 H H H D D 203 H H D H H 204 H H D D H 205 H H D H D 206 H H D D D 207 H D H H H 208 H D H D H 209 H D H H D 210 H D H D D 211 H D D H H 212 H D D D H 213 H D D H D 214 H D D D D 215 D H H H H 216 D H H D H 217 D H H H D 218 D H H D D 219 D H D H H 220 D H D D H 221 D H D H D 222 D H D D D 223 D D H H H 224 D D H D H 225 D D H H D 226 D D H D D 227 D D D H H 228 D D D D H 229 D D D H D 230 D D D D D 231 H H H H H

In some embodiments, the JAK inhibitor is baricitinib, wherein one or more hydrogen atoms are replaced by deuterium atoms, or a pharmaceutically acceptable salt thereof. In some embodiments, the JAK inhibitor is any of the compounds in U.S. Pat. No. 9,540,367 (which is incorporated herein by reference in its entirety), or a pharmaceutically acceptable salt thereof.

In some embodiments, the JAK inhibitor is a compound of Table 3, or a pharmaceutically acceptable salt thereof. The compounds in Table 3 are selective JAK1 inhibitors (selective over JAK2, JAK3, and TYK2).

TABLE 3 Examples of JAK inhibitors Comp. No. Prep. Name Structure 1 US 2011/ 0224190 (Example 1) {1-{1-[3-Fluoro- 2-(trifluoro- methyl)isonico- tinoyl]piperidin- 4-yl}-3-[4-(7H- pyrrolo[2,3-d] pyrimidin-4-yl)- 1H-pyrazol-1-yl] azetidin-3-yl} acetonitrile (itacitinib;; also known as INCB039110)

2 US 2011/ 0224190 (Example 154) 4-{3-(Cyano- methyl)-3-[4- (7H-pyrrolo [2,3-d]pyrimidin- 4-yl)-1H- pyrazol-1-yl] azetidin-1-yl}- N-[4-fluoro-2- (trifluoromethyl) phenyl] piperidine-1- carboxamide

3 US 2011/ 0224190 (Example 85) [3-[4-(7H- pyrrolo[2,3-d] pyrimidin-4-yl)- 1H-pyrazol-1-yl]- 1-(1-{[2- (trifluoromethyl) pyrimidin-4-yl] carbonyl}piperidin- 4-yl)azetidin-3- yl]acetonitrile

4 US 2014/03430 30 (Example 7) 4-[3-(cyanomethyl)- 3-(3′,5′-dimethyl- 1H,1′H-4,4′- bipyrazol-1-yl) azetidin-1-yl] 2,5-difluoro-N- [(1S)-2,2,2- trifluoro-1- methylethyl] benzamide

5 US 2014/01211 98 (Example 20) ((2R,5S)-5-{2- [(1R)-1- hydroxyethyl]- 1H-imidazo [4,5-d]thieno [3,2-b]pyridin- 1-yl}tetrahydro- 2H-pyran-2-yl) acetonitrile

6 US 2010/ 0298334 (Example 2) 3-[1-(6-chloro- pyridin-2-yl) pyrrolidin-3-yl]- 3-[4-(7H- pyrrolo[2,3-d] pyrimidin-4- yl)-1H-pyrazol- 1-yl] propanenitrile

7 US 2010/ 0298334 (Example 13c) 3-(1-[1,3] oxazolo[5,4-b] pyridin-2-yl- pyrrolidin-3-yl)- 3-[4-(7H- pyrrolo[2,3-d] pyrimidin-4-yl)- 1H-pyrazol-1-yl] propanenitrile

8 US 2011/ 0059951 (Example 12) 4-[(4-{3-cyano- 2-[4-(7H- pyrrolo[2,3-d] pyrimidin-4-yl)- 1H-pyrazol-1- yl]propyl} piperazin-1-yl) carbonyl]-3- fluoro- benzonitrile

9 US 2011/ 0059951 (Example 13) 4-[(4-{3-cyano- 2-[3-(7H- pyrrolo[2,3-d] pyrimidin-4-yl)- 1H-pyrrol-1-yl] propyl} piperazin-1-yl) carbonyl]-3- fluoro- benzonitrile

10 US 2012/ 0149681 (Example 7b) [trans-1-[4-(7H- pyrrolo[2,3-d] pyrimidin-4-yl)- 1H-pyrazol-1-yl]- 3-(4-{[2- (trifluoromethyl) pyrimidin-4-yl] carbonyl} piperazin-1-yl) cyclobutyl] acetonitrile

11 US 2012/ 0149681 (Example 157) {trans-3-(4- {[4-[(3- hydroxy- azetidin-1-yl) methyl]-6- (trifluoromethyl) pyridin-2-yl] oxy}piperidin- 1-yl)-1-[4-(7H- pyrrolo[2,3-d] pyrimidin-4-yl)- 1H-pyrazol-1- yl]cyclobutyl} acetonitrile

12 US 2012/ 0149681 (Example 161) {trans-3-(4- {[4-{[(2S)-2- (hydroxymethyl) pyrrolidin-1-yl] methyl}-6- (trifluoromethyl) pyridin-2-yl] oxy}piperidin- 1-yl)-1-[4-(7H- pyrrolo[2,3-d] pyrimidin-4-yl)- 1H-pyrazol-1- yl]cyclobutyl} acetonitrile

13 US 2012/ 0149681 (Example 162) {trans-3-(4- {[4-{[(2R)-2- (hydroxymethyl) pyrrolidin-1-yl] methyl}-6- (trifluoromethyl) pyridin-2-yl] oxy}piperidin- 1-yl)-1-[4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl)- 1H-pyrazol-1-yl] cyclobutyl} acetonitrile

14 US 2012/ 0149682 (Example 20) 4-(4-{3- [(dimethylamino) methyl]-5- fluorophenoxy} piperidin-1-yl)- 3-[4-(7H- pyrrolo[2,3-d] pyrimidin-4-yl)- 1H-pyrazol-1-yl] butanenitrile

15 US 2013/ 0018034 (Example 18) 5-{3-(cyano- methyl)-3-[4- (7H-pyrrolo [2,3-d]pyrimidin- 4-yl)-1H- pyrazol-1-yl] azetidin-1-yl}- N-isopropyl- pyrazine-2- carboxamide

16 US 2013/ 0018034 (Example 28) 4-{3-(cyano- methyl)-3-[4- (7H-pyrrolo [2,3-d]pyrimidin- 4-yl)-1H- pyrazol-1-yl] azetidin-1-yl}- 2,5-difluoro- N-[(1S)-2,2,2- trifluoro-1- methylethyl] benzamide

17 US 2013/ 0018034 (Example 34) 5-{3-(cyano- methyl)-3-[4- (1H-pyrrolo [2,3-b]pyridin- 4-yl)-1H- pyrazol-1-yl] azetidin-1-yl}- N-isopropyl- pyrazine-2- carboxamide

18 US 2013/ 0045963 (Example 45) {1-(cis-4-{[6- (2-hydroxy- ethyl)-2- (trifluoromethyl) pyrimidin-4-yl] oxy}cyclohexyl)- 3-[4-(7H- pyrrolo[2,3-d] pyrimidin-4-yl)- 1H-pyrazol-1- yl]azetidin-3- yl}acetonitrile

19 US 2013/ 0045963 (Example 65) {1-(cis-4-{[4- [(ethylamino) methyl]-6- (trifluoromethyl) pyridin-2-yl] oxy}cyclohexyl)- 3-[4-(7H- pyrrolo[2,3-d] pyrimidin-4-yl)- 1H-pyrazol-1- yl]azetidin-3- yl}acetonitrile

20 US 2013/ 0045963 (Example 69) {1-(cis-4-{[4- (1-hydroxy-1- methylethyl)-6- (trifluoromethyl) pyridin-2-yl] oxy}cyclohexyl)- 3-[4-(7H- pyrrolo[2,3-d] pyrimidin-4-yl)- 1H-pyrazol-1- yl]azetidin-3- yl}acetonitrile

21 US 2013/ 0045963 (Example 95) {1-(cis-4-{[4- {[(3R)-3- hydroxy- pyrrolidin-1- yl]methyl}-6- (trifluoromethyl) pyridin-2-yl] oxy}cyclohexyl)- 3-[4-(7H- pyrrolo[2,3-d] pyrimidin-4-yl)- 1H-pyrazol-1- yl]azetidin-3- yl}acetonitrile

22 US 2013/ 0045963 (Example 95) {1-(cis-4-{[4- {[(3S)-3- hydroxy- pyrrolidin-1-yl] methyl}-6- (trifluoromethyl) pyridin-2-yl] oxy}cyclohexyl)- 3-[4-(7H- pyrrolo[2,3-d] pyrimidin-4-yl)- 1H-pyrazol-1- yl]azetidin-3- yl}acetonitrile

23 US 2014/ 0005166 (Example 1) {trans-3-(4-{[4- ({[(1S)-2- hydroxy-1- methylethyl] amino}methyl)- 6-(trifluoro- methyl) pyridin-2-yl] oxy}piperidin- 1-yl)-1-[4-(7H- pyrrolo[2,3-d] pyrimidin-4-yl)- 1H-pyrazol-1- yl]cyclobutyl} acetonitrile

24 US 2014/ 0005166 (Example 14) {trans-3-(4-{[4- ({[(2R)-2- hydroxypropyl] amino}methyl)- 6-(trifluoro- methyl)pyridin- 2-yl]oxy} piperidin-1-yl)- 1-[4-(7H- pyrrolo[2,3-d] pyrimidin-4-yl)- 1H-pyrazol-1- yl]cyclobutyl} acetonitrile

25 US 2014/ 0005166 (Example 15) {trans-3-(4-{[4- ({[(2S)-2- hydroxypropyl] amino}methyl)- 6-(trifluoro- methyl)pyridin- 2-yl]oxy} piperidin-1-yl)- 1-[4-(7H- pyrrolo[2,3-d] pyrimidin-4-yl)- 1H-pyrazol-1- yl]cyclobutyl} acetonitrile

26 US 2014/ 0005166 (Example 20) {trans-3-(4-{[4- (2-hydroxy- ethyl)-6- (trifluoromethyl) pyridin-2-yl] oxy}piperidin- 1-yl)-1-[4-(7H- pyrrolo[2,3-d] pyrimidin-4-yl)- 1H-pyrazol-1- yl]cyclobutyl} acetonitrile

In some embodiments, the JAK inhibitor is {1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile, or a pharmaceutically acceptable salt thereof.

In some embodiments, the JAK inhibitor is {1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3 [4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile adipic acid salt.

The synthesis and preparation of {1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3 [4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile and the adipic acid salt of the same can be found, e.g., in US Patent Publ. No. 2011/0224190, filed Mar. 9, 2011, US Patent Publ. No. 2013/0060026, filed Sep. 6, 2012, and US Patent Publ. No. 2014/0256941, filed Mar. 5, 2014, each of which is incorporated herein by reference in its entirety.

In some embodiments, the JAK inhibitor is 4-[3-(cyanomethyl)-3-(3′,5′-dimethyl-1H,1′H-4,4′-bipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide, or a pharmaceutically acceptable salt thereof.

In some embodiments, the JAK inhibitor is 4-[3-(cyanomethyl)-3-(3′,5′-dimethyl-1H,1′H-4,4′-bipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide phosphoric acid salt.

The synthesis and preparation of 4-[3-(cyanomethyl)-3-(3′,5′-dimethyl-1H,1′H-4,4′-bipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide and the phosphoric acid salt of the same can be found, e.g., in US Patent Publ. No. US 2014/0343030, filed May 16, 2014, which is incorporated herein by reference in its entirety.

In some embodiments, the JAK inhibitor is ((2R,5S)-5-{2-[(1R)-1-hydroxyethyl]-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl}tetrahydro-2H-pyran-2-yl)acetonitrile, or a pharmaceutically acceptable salt thereof.

In some embodiments, the JAK inhibitor is ((2R,5S)-5-{2-[(1R)-1-hydroxyethyl]-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl}tetrahydro-2H-pyran-2-yl)acetonitrile monohydrate.

Synthesis of ((2R,5S)-5-{2-[(1R)-1-hydroxyethyl]-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl}tetrahydro-2H-pyran-2-yl)acetonitrile and characterization of the anhydrous and monohydrate forms of the same are described in US Patent Publ. No. 2014/0121198, filed Oct. 31, 2013 and US Patent Publ. No. 2015/0344497, filed Apr. 29, 2015, each of which is incorporated herein by reference in its entirety.

In some embodiments, the compounds of Table 3 are prepared by the synthetic procedures described in US Patent Publ. No. 2011/0224190, filed Mar. 9, 2011, US Patent Publ. No. 2014/0343030, filed May 16, 2014, US Patent Publ. No. 2014/0121198, filed Oct. 31, 2013, US Patent Publ. No. 2010/0298334, filed May 21, 2010, US Patent Publ. No. 2011/0059951, filed Aug. 31, 2010, US Patent Publ. No. 2012/0149681, filed Nov. 18, 2011, US Patent Publ. No. 2012/0149682, filed Nov. 18, 2011, US Patent Publ. 2013/0018034, filed Jun. 19, 2012, US Patent Publ. No. 2013/0045963, filed Aug. 17, 2012, and US Patent Publ. No. 2014/0005166, filed May 17, 2013, each of which is incorporated herein by reference in its entirety.

In some embodiments, JAK inhibitor is selected from the compounds, or pharmaceutically acceptable salts thereof, of US Patent Publ. No. 2011/0224190, filed Mar. 9, 2011, US Patent Publ. No. 2014/0343030, filed May 16, 2014, US Patent Publ. No. 2014/0121198, filed Oct. 31, 2013, US Patent Publ. No. 2010/0298334, filed May 21, 2010, US Patent Publ. No. 2011/0059951, filed Aug. 31, 2010, US Patent Publ. No. 2012/0149681, filed Nov. 18, 2011, US Patent Publ. No. 2012/0149682, filed Nov. 18, 2011, US Patent Publ. 2013/0018034, filed Jun. 19, 2012, US Patent Publ. No. 2013/0045963, filed Aug. 17, 2012, and US Patent Publ. No. 2014/0005166, filed May 17, 2013, each of which is incorporated herein by reference in its entirety.

Methods of Treatment

The methods disclosed herein enable the assessment of whether or not a subject having, suspected of having or at risk of developing GvHD is likely to respond (e.g., likely to have greater improvement in disease as evidenced by reduced disease severity and/or disease remission/resolution) to a therapy comprising a JAK inhibitor. A subject having, suspected of having or at risk of developing GvHD who is likely to respond to a JAK inhibitor can be administered a JAK inhibitor (e.g., itacitinib). Conversely, a subject having, suspected of having or at risk of developing GvHD who is less likely to respond to a JAK inhibitor (e.g., itacitinib) can be administered an additional therapy that is suitable for treatment of GvHD.

The methods of this disclosure also enable the stratification of subjects having, suspected of having or at risk of developing GvHD into groups of subjects that are more likely to benefit, and groups of subjects that are less likely to benefit, from treatment comprising a JAK inhibitor. The ability to select such subjects from a pool of GvHD subjects who are being considered for treatment with a JAK inhibitor is beneficial for administering an effective treatment to the subject.

In one embodiment, the subject to be treated with a JAK inhibitor (e.g., itacitinib) has, is suspected of having, or is likely to develop GvHD. In certain embodiments, the subject to be treated with a therapy comprising a JAK inhibitor (e.g., itacitinib) has, is suspected of having, or is likely to develop acute GvHD. In other embodiments, the subject to be treated with a therapy comprising a JAK inhibitor (e.g., itacitinib) has, is suspected of having, or is likely to develop chronic GvHD.

If the subject having GvHD is more likely to respond to a therapy comprising a JAK inhibitor (based on concentrations of one or more of the biomarkers described above (see Tables 1 and 2)), the subject can then be administered an effective amount of the JAK inhibitor (e.g., itacitinib). An effective amount of the JAK inhibitor can suitably be determined by a health care practitioner taking into account, for example, the characteristics of the patient (age, sex, weight, race, etc.), the progression of the disease, and prior exposure to the drug. If the subject is less likely to respond to a therapy comprising a JAK inhibitor, the subject can then be optionally administered a therapy that does not comprise a JAK inhibitor.

The methods can also be applied to individuals at risk of developing GvHD. Such individuals include those who (i) have undergone a transplant (e.g., a hematopoietic stem cell transplant) but have not developed GvHD, or (ii) are preparing for receipt of a transplant (e.g., a hematopoietic stem cell transplant).

After stratifying or selecting a subject based on whether the subject will be more likely or less likely to respond to a JAK inhibitor, a medical practitioner (e.g., a doctor) can administer the appropriate therapeutic modality to the subject. Methods of administering a JAK inhibitor are well known in the art.

In cases where the subject having GvHD and predicted to respond to a JAK inhibitor has been previously administered one or more non-JAK inhibitor therapies, the therapy comprising a JAK inhibitor can replace or augment a previously or currently administered therapy. For example, upon treating with the therapy comprising a JAK inhibitor, administration of the one or more non-JAK inhibitor therapies can cease or diminish, e.g., be administered at lower levels. Administration of the previous therapy can be maintained while the therapy comprising a JAK inhibitor is administered. In some embodiments, a previous therapy can be maintained until the level of the therapy comprising a JAK inhibitor reaches a level sufficient to provide a therapeutic effect.

A subject treated with a JAK inhibitor (e.g., itacitinib) according to the methods described herein can be treated in combination with one or more additional compositions that are effective for treatment of GvHD. Examples of compositions that can be used in such combination treatment include corticosteroids (e.g., methylprednisolone or prednisone), methotrexate, cyclosporine, mycophenolate mofetil, tacrolimus, sirolimus, everolimus, antithymocyte globulin, alemtuzumab, cyclophosphamide, ibrutinib, imatinib, infliximab, etanercept, tocilizumab, alemtuzumab, basiliximab, daclizumab, rituximab, denileukin diftitox, pentostatin, ciclosporin, thalidomide, halofuginone, hydroxychloroquine, and mesenchymal stem cells.

The following are examples of the practice of the invention. They are not to be construed as limiting the scope of the invention in any way.

EXAMPLES Example 1: Identification of Proteins Differentially Expressed in Patients with Acute Graft-Versus-Host Disease that are Complete Responders to Treatment with Itacitinib

Plasma samples were collected from individuals enrolled in a study of itacitinib in combination with corticosteroids for the treatment of Acute Graft-Versus-Host Disease (GvHD). All subjects underwent a first allogeneic hematopoietic stem cell transplantation from any donor source (matched unrelated donor, sibling, haploidentical) using bone marrow, peripheral blood stem cells, or cord blood for hematologic malignancies. The subjects exhibited clinically suspected Grades IIB to IVD acute GvHD, occurring after the allogeneic hematopoietic stem cell transplant. All subjects consented to the blood collection.

Once collected, plasma samples underwent broad proteomic profiling using OLINK™, which allows analysis of >1000 proteins. Samples were separated into the following groups based on the clinical response to treatment with itacitinib (INCB039110). Specifically, samples were classified as “complete responder” (CR), “partial/mixed responder”, or “progressive disease/death” (PD/Death) based on their therapeutic response at day 28 of treatment.

Broad proteomic analysis of plasma identified a total of 118 differentially expressed proteins between the CR and PD/Death groups of subjects. Differentially expressed proteins were those that showed a statistically significant difference (p<0.05) and at least a 1.2 fold change between baselines of complete responders and progressive disease/death cohorts. Fold change in this example represents the change of a baseline protein expression level between complete responders and progressive disease/death groups of subjects. Fifty-three proteins were increased and 65 proteins were decreased in CR compared to PD/Death (Table 4). Down-regulated proteins are proteins whose expression decreased over time, while up-regulated proteins are proteins whose expression increased over time. Fold change in expression is shown for each protein, which is a ratio of protein expression level post-treatment to expression level pre-treatment (baseline). Values greater than 1 indicate an increase from baseline, whereas values less than 1 indicate a decrease from baseline.

TABLE 4 Differentially Expressed Proteins at Baseline in the Plasma of Complete Responders Compared to the Progressive Disease/Death Groups Up-Regulated in CR Compared Down-Regulated in CR Compared to PD/Death to PD/Death Fold Raw Fold Raw Protein Change P Value Protein Change P Value PON3 3.9728 0.0005 MCP-3 −6.0399 0.0004 GCG 3.481 0.0022 CA5A −4.5712 0.0085 SCF 3.0746 0.003 CALCA −4.4035 0.0349 PDGF 3.0721 0.0188 HAOX1 −4.3448 0.0251 subunit B IL8 −4.0642 0.0216 LEP 3.0194 0.0441 IL6 −3.8938 0.0207 FKBP1B 2.9644 0.0357 SPINK1 −3.8821 0.012 MBL2 2.9639 0.0094 CXCL10 −3.8124 0.0065 SCF 2.9526 0.0026 SULT2A1 −3.5143 0.0149 GAL 2.936 0.0119 IL6 −3.4705 0.0258 SCF 2.9112 0.003 ENPP7 −3.3791 0.0388 ITGB1BP2 2.7985 0.0306 PLXNB1 −2.99  0.0105 PVALB 2.6979 0.0084 VAMP5 −2.9842 0.0315 THPO 2.6532 0.0056 CCL19 −2.9578 0.0041 CD40-L 2.645 0.041 CTSL1 −2.9056 0.0018 ANG-1 2.6308 0.0116 ACE2 −2.8742 0.0054 SCGB3A1 2.5332 0.0093 IL6 −2.8377 0.0121 CD69 2.4963 0.0119 CRTAM −2.6741 0.0312 FAM3B 2.481 0.0235 ALDH1A1 −2.6667 0.0141 GH 2.4121 0.0155 SIGLEC10 −2.5993 0.0148 CCL5 2.3402 0.0064 KRT19 −2.5964 0.0312 MANF 2.3026 0.0174 SLAMF8 −2.5417 0.0189 SRC 2.1146 0.036 IL6 −2.5189 0.0141 CRISP2 2.0487 0.0148 CDCP1 −2.4173 0.0113 SAA4 2.0164 0.0348 N2DL-2 −2.3079 0.0075 CR2 2.0089 0.0197 GZMB −2.307  0.0289 SERPINA5 2.0067 0.0044 TNFRSF6B −2.3053 0.0037 PFKM 1.9756 8.93E−05 KYNU −2.2368 0.0217 APOM 1.9231 0.0255 FOSB −2.2367 0.0169 DCTN2 1.8684 0.0304 ALDH3A1 −2.1808 0.0104 HSD11B1 1.8131 0.0173 IGFBP-1 −2.1383 0.0472 PDGF 1.7697 0.0424 CLM-1 −2.1323 0.012 subunit A NFATC3 −2.0978 0.0189 IGFBP3 1.7304 0.0287 HAVCR2 −2.0933 0.0196 HS3ST3B1 1.7175 0.0355 TNF-R2 −2.0374 0.0091 CDSN 1.7055 0.0227 DDAH1 −2.036  0.0289 APP 1.7003 0.0484 CD74 −1.9824 0.0051 TWEAK 1.6972 0.0338 CKAP4 −1.9573 0.0069 TN-R 1.653 0.0321 NINJ1 −1.9125 0.0043 AMBP 1.6302 0.0127 ENTPD2 −1.8643 0.0227 CNTN1 1.5831 0.0275 TNFRSF9 −1.8239 0.0276 GCP5 1.5583 0.0383 SIGLEC1 −1.8208 0.0229 CNDP1 1.555 0.0034 PREB −1.8178 0.0327 NCAM1 1.5413 0.0011 AHCY −1.7883 0.043 PROC 1.514 0.0289 IL12RB1 −1.7236 0.0186 F11 1.489 0.008 TNFRSF10A −1.7188 0.0133 NCAN 1.4147 0.0259 SIRPB1 −1.7175 0.0467 NTRK3 1.3885 0.0255 DSC2 −1.6964 0.0261 TIMP4 1.3827 0.044 U-PAR −1.687  0.0422 VEGFD 1.3649 0.0365 TNFRSF4 −1.677  0.0423 HSP 27 1.3522 0.0446 TNFRSF10A −1.6687 0.0145 GALNT10 1.3452 0.0207 IL-18R1 −1.668  0.0121 CCL11 1.3225 0.0111 IL-1ra −1.6559 0.0334 LY75 1.2722 0.0376 CLEC7A −1.6558 0.0316 DKKL1 1.2291 0.0499 SIGLEC7 −1.5857 0.0148 COL4A1 −1.5794 0.0156 TLR3 −1.5753 0.0412 PD-L1 −1.5245 0.0139 IL-18BP −1.4932 0.0342 PILRA −1.4815 0.0341 CCL15 −1.4614 0.0279 uPA −1.4223 0.0314 DLL1 −1.2888 0.0194 THBS2 −1.2178 0.004 SPON2 −1.1748 0.0105

Example 2: Characterization of Protein Expression During the Course of Treatment

Plasma samples were collected from individuals enrolled in the clinical study of Example 1 at baseline and at day 28. Table 5 lists proteins that were significantly modulated by treatment between baseline and day 28. Table 6 lists proteins that were stably expressed throughout the study and were not significantly modulated by treatment between baseline and day 28.

Table 5 identifies proteins that changed in complete responders between day 1 (baseline) to day 28. Fold change in this example represents the change of a protein level between day 1 (baseline) versus day 28. A paired t test was used to compare the relative change between level of a protein between day 1 and day 28. Table 5 identifies biomarkers of therapeutic response.

TABLE 5 Proteins Significantly Modulated in Complete Responders Between Baseline and Day 28 Increased Expression from Decreased Expression from Baseline to Day 28 Baseline to Day 28 Protein Raw Fold Raw Change Fold P Value Protein Change P Value GCG 2.3961 0.0168 PDGF subunit −2.7388 0.001 GAL 2.0603 0.0052 B THPO 1.8262 0.0094 FKBP1B −2.5654 0.0036 FAM3B 1.7937 0.0011 ITGB1BP2 −2.1159 0.0047 CNDP1 1.5692 0.0134 CD69 −2.1102 0.0016 CCL11 1.401 0.0013 ANG-1 −2.0923 0.0062 SERP1NA5 1.3313 0.0089 PVALB −2.0632 0.0145 DKKL1 1.2705 0.0014 CD40-L −1.9741 0.0256 NCAM1 1.2481 0.0284 CCL5 −1.9502 0.0065 SPON2 1.1003 0.0106 HS3ST3B1 −1.9303 0.0193 THBS2 1.0834 0.0051 MBL2 −1.9284 0.0215 GCG 2.3961 0.0168 CLEC7A −1.8985 0.0006 GAL 2.0603 0.0052 APP −1.8356 0.0053 THPO 1.8262 0.0094 PDGF subunit −1.7507 0.0068 FAM3B 1.7937 0.0011 A CNDP1 1.5692 0.0134 DCTN2 −1.6367 0.0274 CCL11 1.401 0.0013 SLAMF8 −1.5899 0.0244 SERPINA5 1.3313 0.0089 VAMP5 −1.5851 0.0053 DKKL1 1.2705 0.0014 SIGLEC10 −1.4859 0.0226 NCAM1 1.2481 0.0284 CLM-1 −1.4729 0.0319 SPON2 1.1003 0.0106 DSC2 −1.4423 0.0017 THBS2 1.0834 0.0051 HAVCR2 −1.3876 0.0126 SIRPB1 −1.3861 0.0072 COL4A1 −1.3412 0.0369 PILRA −1.2552 0.0282 LY75 −1.2217 0.024

Table 6 identifies proteins that did not modulate in complete responders between baseline (day 1) and day 28. Therefore, these proteins are designated as baseline predictive biomarkers.

TABLE 6 Proteins Stably Expressed in Complete Responders Between Day 1 and Day 28 Proteins Proteins Increased But Not Significant Decreased But Not Significant Fold Raw P Fold Raw Protein Change Value Protein Change P Value IL6 1.7545 0.1382 MANF −1.6753 0.0584 HAOX1 1.7169 0.0995 SRC −1.6394 0.0535 IGFBP-1 1.6812 0.1999 TWEAK −1.4741 0.1325 IL6 1.6701 0.1579 CR2 −1.4025 0.0966 IL6 1.5995 0.1953 SIGLEC1 −1.3628 0.0991 ENPP7 1.5432 0.1385 TNF-R2 −1.3591 0.0931 IL6 1.5407 0.2577 TNFRSF6B −1.3442 0.1025 SCF 1.5147 0.0799 AHCY −1.3275 0.0854 SCGB3A1 1.5084 0.0863 CRTAM −1.3192 0.3574 SCF 1.4346 0.0961 CKAP4 −1.2761 0.1239 SCF 1.3988 0.1184 CA5A −1.267  0.3739 PON3 1.3708 0.0631 CALCA −1.2236 0.6545 MCP-3 1.3171 0.2406 IL-1ra −1.2159 0.3242 IL8 1.286 0.2913 TNFRSF9 −1.2128 0.2866 NINJ1 1.239 0.0994 DDAH1 −1.1849 0.2464 NTRK3 1.2351 0.0846 HSP 27 −1.1778 0.1094 CNTN1 1.2158 0.1831 SAA4 −1.1593 0.5157 ACE2 1.2118 0.4374 PFKM −1.1575 0.2464 IGFBP3 1.1814 0.3669 TIMP4 −1.1371 0.2962 GH 1.1727 0.676 LEP −1.1316 0.6995 CCL15 1.1726 0.2308 IL-18BP −1.1268 0.5792 SULT2A1 1.169 0.6268 CDSN −1.1137 0.2888 uPA 1.1545 0.1665 PD-L1 −1.0969 0.4905 FOSB 1.1542 0.1953 IL-18R1 −1.0806 0.2996 PLXNB1 1.1495 0.1726 N2DL-2 −1.0683 0.695  PREB 1.1356 0.1311 CCL19 −1.061  0.8598 CXCL10 1.133 0.805 ALDH3A1 −1.0553 0.5588 PROC 1.1313 0.4097 U-PAR −1.054  0.6793 TNFRSF4 1.1251 0.4331 SPINK1 −1.038  0.8215 KYNU 1.1183 0.5652 TNFRSF10A −1.0214 0.7961 AMBP 1.1176 0.3079 CTSL1 −1.0209 0.9115 VEGFD 1.1116 0.1912 F11 −1.0199 0.8362 DLL1 1.1055 0.2106 SIGLEC7 −1.015  0.9064 NCAN 1.0964 0.0631 KRT19 −1.0136 0.9496 TN-R 1.0934 0.5608 IL12RB1 −1.0129 0.9484 GALNT10 1.064 0.5285 CRISP2 −1.0113 0.8654 NFATC3 1.0637 0.7243 TNFRSF10A −1.0093 0.9187 CDCP1 1.0579 0.7832 ALDH1A1 −1.0018 0.9927 HSD11B1 1.0468 0.7981 APOM 1.0434 0.8235 ENTPD2 1.0429 0.3761 TLR3 1.035 0.5615 GCP5 1.0249 0.7675 GZMB 1.0134 0.9703 CD74 1.0118 0.8996

Example 3: Identification of Proteins that do or do not Correlate with REG3α, TNFR1, and ST2

Several inflammatory mediators have been identified and associated with increased risk of acute GvHD in steroid treated subjects that have received a hematopoietic stem cell transplant. These inflammatory mediators include REG3α, TNFR1, and ST2 (Hartwell et al., “An early-biomarker algorithm predicts lethal graft-versus-host disease and survival, JCI Insight, 2(3):e89798). Using plasma samples from subjects enrolled in the clinical study of Example 1, proteins were evaluated for their correlation with REG3α, TNFR1, and ST2 levels at baseline. Correlation refers to potential biomarkers showing similar change or distribution as REG3α, TNFR1, and ST2. Table 7 identifies proteins that significantly (p<0.1) correlate with REG3α, TNFR1, and ST2 at baseline. Table 8 identifies proteins that do not significantly (p>0.1) correlate with REG3α, TNFR1, and ST2 at baseline.

TABLE 7 Proteins that Significantly Correlate with REG3α, TNFR1, and ST2 Proteins that correlate Proteins that correlate Proteins that correlate with REG3A with TNFR1 with ST2 Protein Correlation P Value Protein Correlation P Value Protein Correlation P Value CD74 0.9158 0.0002 FAM3B 0.7439 0.0136 HAOX1 0.7127 0.0207 CALCA 0.8675 0.0011 TIMP4 0.7076 0.0221 VAMP5 0.5498 0.0996 N2DL-2 0.8435 0.0022 AMBP 0.6938 0.0261 TLR3 −0.7273 0.0171 SPINK1 0.8116 0.0044 GCG 0.6566 0.0392 HS3ST3B1 −0.6144 0.0588 CKAP4 0.7651 0.0099 MANF 0.621 0.0553 NCAM1 −0.6032 0.0648 NFATC3 0.7568 0.0113 VAMP5 0.6094 0.0614 SCGB3A1 −0.5909 0.072 SLAMF8 0.7158 0.0199 LEP 0.5986 0.0675 DSC2 0.7037 0.0231 IGFBP-1 0.5712 0.0846 PLXNB1 0.701 0.0239 F11 0.5683 0.0865 VEGFD 0.6982 0.0247 PD-L1 0.5512 0.0987 HAVCR2 0.6898 0.0273 ENPP7 −0.5962 0.0689 KRT19 0.6864 0.0284 PILRA 0.6774 0.0314 IL-18BP 0.659 0.0382 TNF-R2 0.6456 0.0438 CDCP1 0.6439 0.0445 U-PAR 0.6349 0.0486 CLEC7A 0.6315 0.0502 CCL15 0.6276 0.0521 IL-1ra 0.6237 0.054 SAA4 0.607 0.0627 IL-18R1 0.6039 0.0645 TNFRSF9 0.5953 0.0694 CLM-1 0.5886 0.0734 ITGB1BP2 0.5683 0.0865 APOM 0.5667 0.0876 IL12RB1 0.5528 0.0975 CRISP2 −0.6654 0.0357

TABLE 8 Proteins that do not Significantly Correlate with REG3α, TNFR1, and ST2 Proteins that do not Proteins that do not Proteins that do not correlate with REG3A correlate with TNFR1 correlate with ST2 Protein Correlation P Value Protein Correlation P Value Protein Correlation P Value CDSN 0.5421 0.1055 SCF 0.5327 0.1129 SRC 0.5154 0.1273 SIRPB1 0.5295 0.1155 SCF 0.5302 0.1149 FOSB 0.4886 0.1519 TNFRSF4 0.5048 0.1368 CCL15 0.5161 0.1267 SULT2A1 0.4758 0.1645 CCL5 0.5026 0.1387 DCTN2 0.5131 0.1293 KYNU 0.4712 0.1692 SIGLEC10 0.5024 0.1389 DDAH1 0.5114 0.1309 SIGLEC10 0.4656 0.1751 SIGLEC7 0.4969 0.144 DLL1 0.5068 0.1349 CTSL1 0.4628 0.1781 MANF 0.4955 0.1453 SIRPB1 0.5053 0.1363 CA5A 0.4588 0.1823 SIGLEC1 0.4867 0.1537 COL4A1 0.5019 0.1393 COL4A1 0.4535 0.188 APP 0.4833 0.1571 SRC 0.4937 0.147 MANF 0.4374 0.2062 CTSL1 0.4797 0.1606 TNFRSF4 0.4851 0.1553 MCP-3 0.4227 0.2236 PDGF 0.4733 0.1671 ALDH3A1 0.4831 0.1572 FAM3B 0.4091 0.2405 subunit A IGFBP-1 0.4368 0.2069 SULT2A1 0.4796 0.1607 GCG 0.4017 0.2499 SPON2 0.4365 0.2072 SERPINA5 0.4683 0.1723 CCL15 0.3789 0.2802 SRC 0.4349 0.209 CLEC7A 0.4674 0.1731 DDAH1 0.3768 0.2832 GCP5 0.4261 0.2196 TNF-R2 0.4671 0.1734 AHCY 0.3758 0.2845 CRTAM 0.42 0.2269 CNTN1 0.4106 0.2386 PD-L1 0.3545 0.3149 TLR3 0.4143 0.2339 FKBP1B 0.404 0.247 SPON2 0.3342 0.3452 PD-L1 0.4068 0.2434 CLM-1 0.39 0.2652 KRT19 0.3166 0.3727 MCP-3 0.4 0.252 SIGLEC1 0.3765 0.2835 CRTAM 0.3123 0.3797 CXCL10 0.3944 0.2594 SCF 0.3748 0.2858 ALDH1A1 0.3079 0.3868 IL8 0.3778 0.2818 MBL2 0.3728 0.2887 ITGB1BP2 0.2858 0.4233 CNTN1 0.3588 0.3086 GALNT10 0.3583 0.3093 NFATC3 0.2854 0.4242 PDGF 0.3581 0.3096 TNFRSF9 0.351 0.3201 IL6 0.2821 0.4298 subunit B FKBP1B 0.3529 0.3172 PILRA 0.3496 0.322 ALDH3A1 0.2716 0.4477 CD69 0.3487 0.3235 CD40-L 0.3423 0.3329 ACE2 0.2692 0.452 ENPP7 0.3255 0.3587 IL-18BP 0.3311 0.3501 IL8 0.2651 0.4591 LY75 0.3251 0.3594 GCP5 0.3298 0.352 IL6 0.2604 0.4675 uPA 0.3224 0.3636 CD69 0.3237 0.3616 DCTN2 0.2494 0.4871 DCTN2 0.3165 0.373 U-PAR 0.3234 0.362 SPINK1 0.2493 0.4872 VAMP5 0.3128 0.3788 TWEAK 0.3205 0.3666 IL6 0.2411 0.5022 ANG-1 0.308 0.3866 DSC2 0.3192 0.3687 CD69 0.2244 0.533 CD40-L 0.3077 0.3871 HSD11B1 0.3159 0.374 VEGFD 0.2227 0.5363 TIMP4 0.302 0.3964 HSP 27 0.2988 0.4018 PLXNB1 0.222 0.5377 CA5A 0.2969 0.4049 NTRK3 0.2821 0.4297 SIRPB1 0.2111 0.5582 TNFRSF6B 0.2891 0.4179 ITGB1BP2 0.2648 0.4597 MBL2 0.2088 0.5627 GCG 0.2604 0.4675 CA5A 0.2477 0.4901 TIMP4 0.2085 0.5633 DDAH1 0.2571 0.4733 SPINK1 0.2403 0.5037 IL6 0.2063 0.5675 KYNU 0.2506 0.485 FOSB 0.2371 0.5096 SIGLEC7 0.2015 0.5767 GH 0.2433 0.4982 IL12RB1 0.22 0.5414 TNF-R2 0.1932 0.5928 IL6 0.2424 0.4998 LY75 0.1997 0.5802 FKBP1B 0.1822 0.6143 MBL2 0.2384 0.5071 CCL19 0.195 0.5893 CNDP1 0.1782 0.6223 TNFRSF10A 0.2273 0.5277 ENTPD2 0.1913 0.5964 GAL 0.1763 0.6262 ACE2 0.222 0.5377 APOM 0.1908 0.5975 DLL1 0.1665 0.6458 IL6 0.2185 0.5442 SPON2 0.1893 0.6004 DSC2 0.1661 0.6464 TNFRSF10A 0.2081 0.564 CKAP4 0.1813 0.6162 SIGLEC1 0.1642 0.6503 IGFBP3 0.2074 0.5653 CCL5 0.1799 0.619 CLEC7A 0.161 0.6568 IL6 0.1987 0.5821 PROC 0.1767 0.6254 HAVCR2 0.1598 0.6592 HSP 27 0.191 0.5972 CTSL1 0.1592 0.6604 U-PAR 0.1579 0.6632 ALDH3A1 0.1869 0.6052 HAVCR2 0.1576 0.6636 CALCA 0.1474 0.6844 CNDP1 0.1755 0.6276 GAL 0.1471 0.6851 CRISP2 0.1461 0.6872 IL6 0.1694 0.64 PON3 0.1353 0.7094 PILRA 0.1439 0.6916 GZMB 0.1604 0.658 TNFRSF10A 0.1328 0.7146 PROC 0.1351 0.7098 SCGB3A1 0.1596 0.6597 CDSN 0.1261 0.7286 NCAN 0.1245 0.7318 CCL19 0.1423 0.6949 TNFRSF6B 0.125 0.7308 PDGF 0.124 0.7328 subunit A ALDH1A1 0.1224 0.7363 TNFRSF10A 0.1009 0.7815 IL-1ra 0.1166 0.7483 FOSB 0.1068 0.769 CRISP2 0.0895 0.8057 LEP 0.1156 0.7504 HAOX1 0.0985 0.7865 KYNU 0.0889 0.8071 GCP5 0.095 0.794 AHCY 0.0951 0.7938 PDGF 0.084 0.8176 SLAMF8 0.0876 0.8099 subunit B NTRK3 0.0842 0.8171 ANG-1 0.079 0.8284 CD74 0.0855 0.8142 FAM3B 0.0842 0.8172 CRTAM 0.0665 0.8552 ENPP7 0.0743 0.8384 COL4A1 0.0703 0.8469 MCP-3 0.0537 0.8828 IGFBP-1 0.0727 0.8419 THPO 0.0603 0.8685 THBS2 0.0536 0.8831 CD40-L 0.0692 0.8494 PROC 0.0564 0.877 SIGLEC7 0.0485 0.8942 TNFRSF10A 0.0606 0.8679 NCAM1 0.0319 0.9304 CR2 0.0378 0.9175 F11 0.0544 0.8813 NCAN 0.0227 0.9504 SAA4 0.0183 0.96 uPA 0.0506 0.8896 PON3 0.0187 0.9591 PREB 0 1 GALNT10 0.0387 0.9154 DLL1 0.0146 0.9681 NINJ1 0 1 THBS2 0.0387 0.9155 NINJ1 0 1 THPO −0.0005 0.9989 CDCP1 0.0266 0.9418 PREB 0 1 PLXNB1 −0.0028 0.9938 PDGF 0.0201 0.956 subunit B SULT2A1 −0.0321 0.9298 NFATC3 −0.0033 0.9928 SCF 0.004 0.9913 TN-R −0.0585 0.8724 PDGF −0.01 0.9781 NTRK3 0.0026 0.9942 subunit A AMBP −0.0823 0.8211 N2DL-2 −0.0164 0.9641 SCF 0.0018 0.9961 GALNT10 −0.0831 0.8194 HAOX1 −0.0276 0.9396 CCL5 0.0015 0.9968 TWEAK −0.1065 0.7697 GZMB −0.0298 0.9349 PREB 0 1 DKKL1 −0.1125 0.7569 uPA −0.03 0.9343 NINJ1 0 1 PVALB −0.118 0.7454 SLAMF8 −0.0371 0.919 TNFRSF10A −0.0047 0.9896 PFKM −0.1796 0.6196 CDCP1 −0.0431 0.9058 TNFRSF9 −0.0047 0.9896 HSD11B1 −0.1829 0.6131 PVALB −0.0674 0.8533 CLM-1 −0.0066 0.9855 ENTPD2 −0.1832 0.6124 CXCL10 −0.0675 0.853 CDSN −0.0137 0.97 HS3ST3B1 −0.2478 0.49 SIGLEC10 −0.1138 0.7543 IL-18BP −0.0184 0.9598 F11 −0.249 0.4879 IL-1ra −0.1167 0.7481 CCL19 −0.0194 0.9576 CR2 −0.2631 0.4626 IGFBP3 −0.1316 0.7171 N2DL-2 −0.0203 0.9556 GAL −0.2958 0.4067 CCL11 −0.1433 0.6928 TNFRSF4 −0.0231 0.9495 SCF −0.3195 0.3681 CALCA −0.1562 0.6666 SCF −0.0345 0.9246 SCF −0.3198 0.3677 NCAN −0.1632 0.6523 HSD11B1 −0.0653 0.8578 SERPINA5 −0.3358 0.3427 TN-R −0.1766 0.6254 APOM −0.0674 0.8532 SCF −0.3513 0.3196 PFKM −0.1914 0.5963 CCL11 −0.0699 0.8479 THBS2 −0.3725 0.2892 DKKL1 −0.2137 0.5532 GZMB −0.0739 0.8393 LEP −0.3739 0.2872 CD74 −0.2144 0.5519 CKAP4 −0.0951 0.7938 CCL11 −0.4076 0.2423 ALDH1A1 −0.2245 0.5329 AMBP −0.122 0.7371 SCGB3A1 −0.2297 0.5233 THPO −0.1323 0.7156 NCAM1 −0.2349 0.5135 LY75 −0.1404 0.6989 IL6 −0.249 0.4878 IL-18R1 −0.1526 0.6738 CNDP1 −0.2571 0.4734 ENTPD2 −0.1561 0.6667 IL6 −0.2641 0.4609 ANG-1 −0.1586 0.6618 IL6 −0.2755 0.441 APP −0.1633 0.6521 KRT19 −0.278 0.4368 IL12RB1 −0.1745 0.6296 ACE2 −0.2926 0.4119 SAA4 −0.1783 0.6221 IL-18R1 −0.3026 0.3954 SERPINA5 −0.1971 0.5852 APP −0.3215 0.365 HSP 27 −0.2018 0.5762 TLR3 −0.3408 0.3352 TN-R −0.204 0.5719 AHCY −0.353 0.317 PFKM −0.2317 0.5196 IL6 −0.3819 0.2762 TNFRSF6B −0.2566 0.4742 VEGFD −0.4069 0.2432 PON3 −0.2767 0.4389 IL8 −0.4509 0.1909 IGFBP3 −0.2965 0.4054 HS3ST3B1 −0.4519 0.1898 PVALB −0.306 0.3899 GH −0.5459 0.1026 DKKL1 −0.3744 0.2865 CNTN1 −0.3974 0.2555 CXCL10 −0.4139 0.2344 GH −0.4141 0.2342 TWEAK −0.471 0.1695 CR2 −0.5445 0.1036

Example 4: Selection of Proteins Capable of Predicting Positive Therapeutic Response with JAK Inhibition in GvHD

Data from the previous examples identified several proteins at baseline that predict a positive therapeutic response, as evidenced by classification as CR at day 28. Proteins listed in (but not limited to) Table 9 were found to be: (1) differentially expressed between the CR and PD/Death treatment groups; (2) stable between baseline and day 28; and (3) not correlated with REG3α, TNFR1, and ST2.

TABLE 9 Proteins Differentially Expressed Between the CR and PD/Death Treatment Groups, Stable Between Baseline and Day 28, and not Correlated with REG3α, TNFR1, and ST2 Proteins Increased in CR Proteins Decreased in CR Fold Raw P Fold Raw P Protein Change Value Protein Change Value PON3 3.9728 0.0005 MCP-3 −6.0399 0.0004 SCF 3.0746 0.003 CA5A −4.5712 0.0085 SCF 2.9526 0.0026 IL8 −4.0642 0.0216 SCF 2.9112 0.003 CXCL10 −3.8124 0.0065 GH 2.4121 0.0155 SULT2A1 −3.5143 0.0149 SRC 2.1146 0.036 IL6 −3.4705 0.0258 CR2 2.0089 0.0197 CCL19 −2.9578 0.0041 PFKM 1.9756 8.93E−05 CTSL1 −2.9056 0.0018 HSD11B1 1.8131 0.0173 ACE2 −2.8742 0.0054 IGFBP3 1.7304 0.0287 IL6 −2.8377 0.0121 CDSN 1.7055 0.0227 CRTAM −2.6741 0.0312 TWEAK 1.6972 0.0338 ALDH1A1 −2.6667 0.0141 TN-R 1.653 0.0321 IL6 −2.5189 0.0141 CNTN1 1.5831 0.0275 GZMB −2.307 0.0289 GCP5 1.5583 0.0383 TNFRSF6B −2.3053 0.0037 PROC 1.514 0.0289 KYNU −2.2368 0.0217 NCAN 1.4147 0.0259 FOSB −2.2367 0.0169 NTRK3 1.3885 0.0255 ALDH3A1 −2.1808 0.0104 HSP 27 1.3522 0.0446 DDAH1 −2.036 0.0289 GALNT10 1.3452 0.0207 NINJA −1.9125 0.0043 ENTPD2 −1.8643 0.0227 SIGLEC1 −1.8208 0.0229 PREB −1.8178 0.0327 AHCY −1.7883 0.043 TNFRSF10A −1.7188 0.0133 TNFRSF4 −1.677 0.0423 SIGLEC7 −1.5857 0.0148 uPA −1.4223 0.0314 DLL1 −1.2888 0.0194

Using a more stringent cutoff of 2 (absolute number), the number of predictive proteins was further reduced (Table 10).

TABLE 10 Selected Proteins Capable of Predicting Positive Therapeutic Response to JAK Inhibition in GvHD Protein Fold Change CR vs PD/Death Raw P Value PON3 3.9728 0.0005 SCF 3.0746 0.003 SCF 2.9526 0.0026 SCF 2.9112 0.003 GH 2.4121 0.0155 SRC 2.1146 0.036 CR2 2.0089 0.0197 MCP-3 −6.0399 0.0004 CA5A −4.5712 0.0085 IL8 −4.0642 0.0216 CXCL10 −3.8124 0.0065 IL6 −3.4705 0.0258 CCL19 −2.9578 0.0041 CTSL1 −2.9056 0.0018 ACE2 −2.8742 0.0054 IL6 −2.8377 0.0121 ALDH1A1 −2.6667 0.0141 IL6 −2.5189 0.0141 TNFRSF6B −2.3053 0.0037 KYNU −2.2368 0.0217 FOSB −2.2367 0.0169 ALDH3A1 −2.1808 0.0104 DDAH1 −2.036 0.0289

Example 5: Identification of Proteins Differentially Expressed in Patients with Acute Graft-Versus-Host Disease that are Complete Responders to Treatment with Itacitinib

The combination of itacitinib with corticosteroids was evaluated in a parallel-cohort phase 1 trial and improved overall responses for both steroid naïve and refractory aGvHD patients. A broad proteomic analysis identified predictive, prognostic, and pharmacodynamic biomarkers of response to the combination treatment.

Ten steroid-naive and eighteen steroid-refractory subjects with aGvHD were enrolled in the clinical trial. Plasma samples were collected from all 28 subjects prior to treatment (screening/baseline) and at day 28 following treatment. All subjects provided written consent prior to enrollment and sample collection. Based on the Center for International Blood and Marrow Transplant Research (CIBMTR) response criteria at day 28, subjects were separated into responders and non-responders. Responders included complete responders (CR; n=10), very good partial responders (VGPR; n=1), and partial responders (PR; n=8). Non-responders included mixed responders (n=2) and progressive disease/death (PD/Death; n=7).

Subjects were treated with corticosteroids in combination with either 200 mg (N=14) or 300 mg (N=14) of itacitinib once daily (QD). Clinical response was not significantly different between the two itacitinib doses; therefore, data from both cohorts were combined. Due to the limited sample size, steroid-naive (N=10) and steroid-refractory (N=18) patients were combined for further analysis. Broad proteomic analysis of over 1000 proteins was conducted by OLINK Proteomics (Watertown, Mass.) using a proximity extension assay as described by the manufacturer. Data are presented as normalized protein expression (NPX) in log 2 scale. Statistical differences were evaluated using unpaired and paired t tests, one-way analysis of covariance (ANOVA), and Pearson Correlation. Significance was conferred when P<0.05.

Proteins were identified based on significant differences between the complete responder and progressive disease/death cohorts at baseline that achieved at least a 1.2 fold change between cohorts. See Table 11. Because some patients were re-classified based on their day 28 response, Table 11 represents an updated list of proteins originally shown in Example 1, Table 4.

A total of 146 differentially expressed proteins between the CR and PD/Death groups of subjects were identified. Fifty-seven proteins were increased and 89 proteins were decreased in CR compared to PD/Death. See Table 11.

TABLE 11 Differentially Expressed Proteins at Baseline in the Plasma of Complete Responders Compared to the Progressive Disease/Death Groups Up-Regulated in CR Compared to Down-Regulated in CR Compared to PD/Death PD/Death Fold Raw P Protein Change Raw P Value Protein Fold Change Value CCL17 6.4545 0.0014 MCP-3 −7.865 0.000012941 PON3 4.8594 0.0005 HAOX1 −7.846 0.0027 LEP 4.402 0.0191 CA5A −7.4693 0.001 GCG 3.904 0.0031 CALCA −5.4107 0.0339 MBL2 3.7419 0.0014 IL8 −5.0031 0.0006 SCF 3.5978 0.0006 AREG −4.8443 0.0377 SCF 3.5041 0.0008 SULT2A1 −4.7009 0.0062 SCF 3.2594 0.0024 VAMP5 −4.3186 0.0064 SCGB3A1 3.1352 0.0051 SPINK1 −4.2632 0.0171 GAL 3.0034 0.0179 SIGLEC10 −3.8434 0.0002 FAM3B 2.8141 0.0165 ENPP7 −3.804 0.032 THPO 2.7391 0.007 ACE2 −3.7433 0.0021 GH 2.6866 0.0085 CTSL1 −3.7378 0.0004 PVALB 2.6266 0.0194 PRSS2 −3.6787 0.0454 ANG-1 2.3398 0.0462 CXCL10 −3.5343 0.0192 GDF-8 2.2804 0.0377 NEP −3.4607 0.033 EN-RAGE 2.2642 0.0432 MFGE8 −3.4384 0.014 CRISP2 2.2475 0.0149 KRT19 −3.2722 0.0153 CR2 2.1595 0.0321 SLAMF8 −3.2642 0.0053 CCL5 2.1066 0.0342 CRTAM −3.2086 0.0232 SERPINA5 2.0891 0.0079 IL6 −3.1755 0.0065 IGFBP3 1.9044 0.0219 ALDH1A1 −3.168 0.0096 PFKM 1.8788 0.0007 CES1 −2.9707 0.0286 TN-R 1.8732 0.0227 REG3A −2.9432 0.0499 KLK6 1.8647 0.0002 KYNU −2.9203 0.0061 AMBP 1.8381 0.003 IL-4RA −2.8507 0.0018 SCGB3A2 1.8338 0.029 CDCP1 −2.792 0.0097 TWEAK 1.8183 0.0426 IL6 −2.781 0.0086 FAM19A5 1.813 0.0186 IL6 −2.7351 0.0088 CNTN1 1.791 0.0131 MVK −2.6813 0.0346 VWC2 1.7842 0.0316 FOSB −2.6284 0.0075 CD207 1.7751 0.0153 NFATC3 −2.5865 0.0042 HSD11B1 1.7507 0.0446 N2DL-2 −2.5399 0.0032 KIT 1.7369 0.0439 IL6 −2.5099 0.0151 Notch 3 1.7306 0.0281 DDAH1 −2.5062 0.0089 GCP5 1.7126 0.0062 IGFBP-1 −2.4965 0.0315 BCAN 1.6911 0.0092 ALDH3A1 −2.477 0.003 CDSN 1.6718 0.0496 CXADR −2.4672 0.0111 hK14 1.6607 0.0429 HAVCR2 −2.4468 0.0022 DRAXIN 1.6547 0.0226 CKAP4 −2.3793 0.0008 NCAM1 1.6306 0.0012 PLXNB1 −2.3572 0.0015 F11 1.5723 0.0109 NINJA −2.3018 0.0004 CNDP1 1.5685 0.0117 TNFRSF6B −2.24 0.0093 TIMP4 1.5269 0.0151 CLM-1 −2.2069 0.0188 NCAN 1.5261 0.0216 CD74 −2.2017 0.0012 WNT9A 1.5125 0.0206 ENTPD2 −2.1935 0.0084 MFAP5 1.4911 0.0342 PREB −2.1563 0.0115 CCL28 1.4765 0.0365 CCL19 −2.1313 0.0252 GALNT10 1.4483 0.0079 SIGLEC1 −2.0772 0.0067 VEGFD 1.4176 0.0439 Gal-4 −2.0631 0.0322 DNER 1.4054 0.0481 HNMT −2.0614 0.0081 CRH 1.4003 0.0285 HTRA2 −2.0308 0.002 CCL11 1.3644 0.0126 VSIG4 −2.0226 0.0258 PAM 1.3347 0.0078 IL-1RT2 −2.0162 0.013 LY75 1.3276 0.0287 TNF-R2 −2.0135 0.0104 CCL11 1.3163 0.0439 IL-18R1 −2.0003 0.0005 KLK10 1.2844 0.0448 SIRPB1 −1.9684 0.02 TNFRSF10A −1.9427 0.0055 AHCY −1.8767 0.038 DSC2 −1.8695 0.0132 IL12RB1 −1.8353 0.0184 TNFRSF10A −1.8332 0.0075 LILRB4 −1.832 0.0435 TRAIL-R2 −1.8307 0.0191 EPHA2 −1.7981 0.0105 U-PAR −1.7842 0.0224 LAIR1 −1.7352 0.0179 SEMA4C −1.7209 0.0178 CLEC7A −1.7189 0.0411 ANGPTL4 −1.7097 0.0494 RTN4R −1.6966 0.0441 CD163 −1.6749 0.013 VCAM1 −1.6662 0.0161 COL4A1 −1.663 0.0168 PDCD1 −1.6537 0.0108 IFN-gamma- −1.6515 0.0152 R1 IL-1ra −1.6115 0.021 CCL15 −1.6016 0.0159 TGM2 −1.5838 0.0286 DAG1 −1.58 0.0345 NECTIN2 −1.5594 0.049 PILRA −1.5248 0.0281 PD-L1 −1.5023 0.0147 SIGLEC7 −1.4749 0.0364 PRDX6 −1.4644 0.0461 DLL1 −1.4438 0.0003 EDIL3 −1.2737 0.0246 THBS2 −1.2257 0.0104 SPON2 −1.212 0.0024

A total of 89 proteins from Table 11 were identified that did not modulate in complete responders between baseline (Day 1) and Day 28. Table 12 represents an updated list of proteins originally shown in Example 2, Table 6. These proteins are designated as baseline predictive biomarkers.

TABLE 12 Proteins from Table 11 that were Stably Expressed in Complete Responders Between Days 1 and 28 Up-Regulated in CR Compared Down-Regulated in CR Compared to PD/Death to PD/Death Fold Fold Change Raw P Change Raw P (D1 vs Value (D1 (D1 vs Value (D1 Protein D28) vs D28 Protein D28) vs D28 PON3   1.3014 0.0529 IL8 1.6324 0.0814 CNTN1   1.1978 0.0872 HAOX1 1.5747 0.09 IGFBP3   1.1493 0.1918 ENPP7 1.4178 0.1392 LEP   1.133  0.5306 ACE2 1.301 0.072 Notch 3   1.1259 0.1626 SULT2A1 1.2251 0.42 TN-R   1.1234 0.2461 MCP-3 1.1945 0.3631 HSD11B1   1.1146 0.3215 CES1 1.1821 0.2881 FAM19A5   1.0841 0.4593 MFGE8 1.1591 0.3239 NCAN   1.0838 0.1333 PLXNB1 1.1347 0.069 F11   1.0834 0.2439 TNFRSF10A 1.1052 0.2863 GDF-8   1.0733 0.6406 CCL15 1.1003 0.3286 CCL28   1.0625 0.2987 TNFRSF10A 1.0972 0.2337 GALNT10   1.0608 0.3706 SEMA4C 1.079 0.4031 BCAN   1.0439 0.6251 PREB 1.0663 0.2 TIMP4   1.0265 0.8344 NFATC 3 1.0623 0.7203 CRISP2   1.0244 0.7055 CCL19 1.0575 0.9296 CD207   1.0177 0.9042 DLL1 1.0479 0.5388 WNT9A   1.0058 0.9334 ENTPD2 1.0197 0.7027 MBL2 −1.439  0.0693 IL-4RA 1.0166 0.8889 EN-RAGE −1.3322 0.1832 EPHA2 1.0139 0.8348 TWEAK −1.2551 0.1314 FOSB 1.0051 0.9581 CR2 −1.1488 0.312 CXCL10 −1.6949 0.2429 MFAP5 −1.1215 0.1239 VAMP5 −1.3344 0.0888 KIT −1.0943 0.3754 ALDH3A1 −1.292 0.1504 GH −1.0614 0.7936 MVK −1.2699 0.0932 PFKM −1.052  0.5545 IL12RB1 −1.2335 0.1735 CDSN −1.05  0.6182 CALCA −1.2293 0.4153 CRH −1.0445 0.6559 AHCY −1.1994 0.1462 GCP5 −1.0404 0.6364 PRSS2 −1.1946 0.2577 KLK6 −1.0386 0.6672 LILRB4 −1.1845 0.114 DRAXIN −1.0356 0.6586 DDAH1 −1.1714 0.1479 IL-1ra −1.1696 0.2023 NECTIN2 −1.1579 0.1167 PDCD1 −1.1485 0.0783 CD74 −1.1483 0.1266 PD-L1 −1.1361 0.181 REG3A −1.1314 0.1677 CA5A −1.1295 0.5551 N2DL-2 −1.1284 0.2413 CDCP1 −1.1249 0.5025 U-PAR −1.0962 0.2869 SIGLEC7 −1.0923 0.3111 ANGPTL4 −1.088 0.618 ALDH1A1 −1.0791 0.6027 SPINK1 −1.0709 0.7767 HTRA2 −1.0707 0.4567 PRDX6 −1.061 0.5849 IL-1RT2 −1.0504 0.6889 IGFBP-1 −1.0455 0.8818 HNMT −1.0338 0.5282 TRAIL-R2 −1.0337 0.6738 CXADR −1.0305 0.484 CTSL1 −1.0288 0.8442 IFN-gamma- −1.0268 0.6494 R1 IL-18R1 −1.0192 0.5615 KRT19 −1.0142 0.9196 KYNU −1.0138 0.9268 TGM2 −1.0122 0.9074

Example 6: Characterization of Protein Expression During the Course of Treatment

Longitudinal differences in protein expression were analyzed by evaluating plasma samples from baseline/screening and day 28. Proteins were identified that were significantly modulated by treatment between screening/baseline and day 28 in responders, including CR, VGPR, and PR patients (N=19). A total of 353 proteins were identified, and are shown in Table 13. From this list, 105 proteins were significantly elevated, and 248 proteins were significantly reduced between baseline and day 28. The list of proteins in Table 13 includes proteins included in Example 2, Table 5, and includes proteins modulated by treatment in CR, VGPR, and PR patients. Table 13 identifies biomarkers of therapeutic response.

TABLE 13 Proteins Significantly Modulated in Responders (CR, VGPR, PR; n = 19) Between Baseline and Day 28 Increased Expression from Decreased Expression from Baseline to Baseline to Day 28 Day 28 Fold Raw P Protein Change Raw P Value Protein Fold Change Value TMPRSS15 3.0633 7.04E−07 INPPL1 4.7131 8.25E−06 CCL11 1.3146 4.74E−06 LAT2 −1.7755 1.01E−05 FAM3B 1.7951 1.23E−05 CLEC7A −1.8103 1.39E−05 MMP7 1.3986 2.15E−05 PPP1R9B −1.6445 2.13E−05 NCAM1 1.3216 9.44E−05 NEMO −1.7984 2.56E−05 Gal-3 1.3504 0.0001 SH2B3 4.7273 3.46E−05 CCL25 2.106 0.0001 BCR −1.8219 5.51E−05 THPO 1.867 0.0002 CD5 −1.9942 6.02E−05 CCL11 1.2818 0.0003 DNAJB1 −1.7332 8.26E−05 hK14 1.5574 0.0004 CCL17 −3.059 8.32E−05 KIM1 1.7741 0.0004 ITGB2 −1.6877 8.97E−05 Flt3L 2.3423 0.0004 BANK1 −2.0084 0.0001 PLIN1 2.4338 0.0005 TPSAB 1 −1.9835 0.0001 SPON2 1.095 0.0006 YES1 −1.9824 0.0001 Gal-4 1.4274 0.0006 LAMP3 −1.7705 0.0001 FABP4 1.6926 0.0006 GM-CSF-R- −1.4489 0.0001 DNER 1.2957 0.0007 alpha GAL 1.6888 0.0008 CNTNAP2 −1.3091 0.0001 KIM1 1.757 0.0009 ZBTB16 −1.9166 0.0002 CPM 1.1479 0.0011 CD163 −1.7206 0.0002 VWC2 1.3331 0.0011 TXLNA −1.5277 0.0002 PPY 1.9703 0.0012 MEPE −1.4941 0.0002 PAM 1.1653 0.0014 BACH1 −1.4794 0.0002 PVR 1.1964 0.0015 MAX −1.449 0.0002 SERPINA5 1.2683 0.0015 NFKBIE −1.2885 0.0002 ST3GAL1 1.3415 0.0016 hOSCAR −1.2603 0.0002 CST5 1.3521 0.002 LAT −2.049 0.0003 CES2 1.4212 0.0022 PTPRJ −1.7421 0.0003 CNDP1 1.3856 0.0024 SIRT2 −1.6014 0.0003 CX3CL1 1.5454 0.0024 SIRPB1 −1.3991 0.0003 HO-1 1.3293 0.0028 AXIN1 −1.8046 0.0004 PRELP 1.1833 0.0029 EIF4G1 −1.5969 0.0004 ADM 1.2035 0.003 PTX3 −1.5567 0.0004 VSIG2 1.2163 0.0031 TRIM5 −1.4743 0.0004 FABP2 3.5893 0.0031 IDUA −1.3887 0.0004 CEACAM5 1.6422 0.0039 NCF2 −2.4951 0.0005 SLITRK2 1.2986 0.004 SELP −1.9244 0.0005 MCP-1 1.6302 0.0044 ARHGEF12 −1.8347 0.0005 NTRK3 1.2526 0.0045 CASP-3 −1.787 0.0005 CLUL1 1.3108 0.0046 CD27 −1.6873 0.0005 CXCL16 1.2028 0.0053 MAP4K5 −1.658 0.0005 SCF 1.4714 0.0056 DAPP1 −1.3805 0.0005 TMPRSS5 1.3307 0.0057 PRDX5 −2.0131 0.0006 REG4 2.1118 0.0059 TLT-2 −1.8372 0.0006 hK11 1.2975 0.0061 PARK7 −1.4104 0.0006 SCF 1.3943 0.0061 IL2-RA −2.2452 0.0007 SCGB3A1 1.5691 0.0061 FOXO1 −1.3557 0.0007 DKKL1 1.1495 0.0071 ST1A1 −1.9523 0.0008 NEP 1.8376 0.0077 GRAP2 −1.6468 0.0008 CPA2 1.7185 0.0088 NBN −1.5879 0.0008 Ep-CAM 1.403 0.0089 CD93 −1.3029 0.0008 THBS2 1.0963 0.0091 FCGR2A −1.5278 0.0009 GPNMB 1.2117 0.0092 DCTN1 −1.4726 0.0009 ITGB5 1.229 0.0104 IRF9 −1.4384 0.0009 GT 1.8699 0.0104 HAVCR2 −1.312 0.0009 APLP1 1.4933 0.0117 CD84 −1.7208 0.001 TACSTD2 1.1931 0.0119 STX8 −1.4796 0.001 NINJA 1.2723 0.012 LY9 −1.4447 0.001 SCF 1.4058 0.0123 ZBTB16 −1.9288 0.0011 REN 1.4074 0.0137 CD200R1 −1.4752 0.0011 GCG 1.8922 0.0137 TOP2B −1.7634 0.0012 SERPINA9 1.5418 0.0151 THY 1 −1.2913 0.0012 KAZALD1 1.2609 0.0154 PRKRA −1.2761 0.0012 SERPINA12 1.567 0.0155 ITGB1BP2 −1.8787 0.0013 PODXL 1.2014 0.0163 CD48 −1.6022 0.0013 AMN 1.2517 0.017 CD244 −1.5176 0.0014 IGF1R 1.2432 0.0171 HCLS1 −1.455 0.0014 LTBP2 1.1874 0.0175 MPO −1.8431 0.0015 ANGPTL3 1.2673 0.0177 SIT1 −1.5501 0.0015 SCARA5 1.1342 0.0179 ICAM3 −1.464 0.0015 B4GAT1 1.2795 0.0179 SOST −1.3214 0.0015 ROBO2 1.249 0.0181 DDX58 −1.6381 0.0016 PDGFC 1.223 0.0199 TNF-R2 −1.5017 0.0016 CA12 1.247 0.0199 TRAF2 −1.4472 0.0016 DDC 1.5485 0.0203 SMAD1 −1.3807 0.0016 EDIL3 1.12 0.0237 LAIR-2 −1.8117 0.0017 XPNPEP2 1.285 0.0268 PIK3AP1 −1.7193 0.0018 PRTG 1.1026 0.0278 VSIG4 −1.5046 0.0018 NQO2 1.0895 0.0282 SIGLEC10 −1.4974 0.0019 AMBP 1.1635 0.0282 CD6 −1.758 0.002 ERBB2 1.1968 0.0283 SKAP1 −1.8075 0.0021 IL6 2.0047 0.0286 FCRL5 −1.3113 0.0021 IL6 1.8649 0.0297 CD177 −1.768 0.0022 MCP-1 1.4322 0.0301 KLRD1 −1.8117 0.0023 VEGFD 1.147 0.0314 ERBB2IP −1.7337 0.0023 GDF-2 1.3656 0.0326 MILR1 −1.3829 0.0023 MUC-16 1.6356 0.0334 MIF −1.7486 0.0024 KLK10 1.2102 0.0341 SNAP23 −1.5751 0.0024 FAM3C 1.3109 0.0341 NUB1 −1.4966 0.0025 uPA 1.1411 0.0346 TIGAR −1.3733 0.0026 IL6 1.7278 0.0347 STAMPB −1.3721 0.0026 AGR2 1.4472 0.0376 DSC2 −1.3652 0.0028 METRNL 1.2013 0.039 LAIR1 −1.3173 0.0028 RTN4R 1.195 0.0391 FKBP1B −1.9994 0.0029 IGF2R 1.1734 0.0395 RASSF2 −1.5477 0.003 NTRK2 1.118 0.0399 FATC1 −1.5044 0.0031 ITGB6 1.152 0.0422 CBL −1.7183 0.0033 SCARF2 1.1639 0.0422 IgG Fc receptor −1.3893 0.0033 SCGB3A2 1.3677 0.0439 II-b RGMB 1.1254 0.0449 GLO1 −1.2571 0.0034 EZR 1.1031 0.0454 PVALB −2.0291 0.0035 PROC 1.243 0.0456 SCAMP3 −1.7405 0.0035 FURIN 1.2365 0.0464 SLAMF8 −1.492 0.0035 PIgR 1.1476 0.049 STX16 −1.4673 0.0035 SMOC2 1.2842 0.0494 TNF-R1 −1.3972 0.0035 DFFA −1.31 0.0038 PPP1R2 −1.3339 0.0039 ANG-1 −1.7898 0.004 CCL5 −1.6357 0.0044 MAP2K6 −1.8184 0.0046 CRKL −1.8003 0.0047 CD38 −1.4181 0.0048 CXCL5 −1.7254 0.0052 PILRA −1.2582 0.0052 IRAK1 −1.2986 0.0053 CA13 −1.8816 0.0054 STX6 −1.4715 0.0055 PRTN3 −1.7658 0.0056 IL-5R-alpha −1.6599 0.0058 ESM-1 −1.4178 0.0058 EGLN1 −1.3184 0.0062 CLEC1B −1.7033 0.0063 TYMP −1.7313 0.0066 SNAP29 −1.6325 0.0067 PDGF subunit A −1.6021 0.0069 TNFRSF11A −1.3519 0.007 gal-8 −1.3154 0.007 GCNT1 −1.3034 0.0071 STK4 −1.8393 0.0072 TNC −1.6915 0.0073 THBS4 −1.7307 0.0075 CLEC4D −1.7084 0.0076 SIGLEC6 −1.9024 0.0078 WASF1 −1.5354 0.0078 WAS −2.133 0.0079 COMT −1.4304 0.0082 RETN −1.8687 0.0084 SH2D1A −1.1574 0.0084 RNASE3 −2.6612 0.0087 PAR-1 −1.2074 0.0088 CD69 −1.7621 0.0089 SIGLEC1 −1.3842 0.0089 FR-gamma −1.2115 0.009 ADAM8 −1.3896 0.0091 AZU1 −2.0976 0.0093 AREG −1.5881 0.0093 SDC4 −1.4678 0.0094 DCTN2 −1.5624 0.0096 BID −1.382 0.0097 RELT −1.3317 0.0099 CLEC5A −1.3618 0.0102 APEX1 −1.5431 0.0103 PSP-D −1.2426 0.0106 FGR −1.4406 0.0108 SELE −1.5291 0.0112 SELL −1.4428 0.0112 MESDC2 −1.7056 0.0114 IQGAP2 −1.5317 0.012 AREG −1.5142 0.0121 CRTAM −1.5805 0.0124 LILRB2 −1.2555 0.0126 TANK −1.3124 0.0127 CPXM1 −1.4779 0.0131 ARSB −1.3432 0.0131 SLAMF1 −1.2218 0.0133 PEBP1 −1.307 0.0135 STIP1 −1.2812 0.0144 PDGF subunit B −1.9124 0.0145 SCARF1 −1.3509 0.0146 DEFA1 −1.9173 0.0148 EPHB4 −1.2339 0.015 ARHGAP1 −1.6039 0.0155 CLM-1 −1.3921 0.0156 DAB2 −1.2548 0.0158 LYN −1.2337 0.0158 CASP-8 −1.4795 0.016 APBB1IP −1.4021 0.0161 ANXA11 −1.3456 0.0167 ICAM1 −1.354 0.017 PRKCQ −1.3251 0.0171 VCAM1 −1.2102 0.0173 HDGF −1.3392 0.0174 CD2AP −1.3188 0.0175 TNFRSF6B −1.3504 0.0177 CLEC1A −1.2841 0.0179 TNFRSF14 −1.2658 0.0179 TACC3 −1.7676 0.0181 MMP-1 −1.4112 0.0186 NRP1 −1.1237 0.0187 ZBTB17 −1.2333 0.0189 NADK −1.3493 0.019 PLXNA4 −1.405 0.0193 MMP-9 −1.9306 0.0198 NCR1 −1.3726 0.0202 AMIGO2 −1.1962 0.0202 FES −1.4934 0.0204 CD79B −1.2372 0.0206 TNXB −1.156 0.0216 TXNDC5 −1.4081 0.0217 TRANCE −1.4034 0.0222 ARG1 −1.3036 0.0225 PCDH17 −1.232 0.0228 LRMP −1.6365 0.0231 C1QTNF1 −1.2979 0.0231 CLM-6 −1.1356 0.0232 CKAP4 −1.1904 0.0237 APP −1.5208 0.0244 PGLYRP1 −1.6181 0.0255 LILRA5 −1.342 0.0271 CLEC10A −1.274 0.028 NMNAT1 −1.4212 0.0286 IL-6RA −1.1901 0.0287 ATG4A −1.3651 0.0289 TIMP1 −1.2337 0.029 COCH −1.22 0.0294 DKN1A −1.4302 0.0303 CD1C −1.5651 0.0305 DECR1 −1.4327 0.0316 DAG1 −1.2406 0.0317 IGFBP-2 −1.2058 0.0321 RET −1.4592 0.0329 GSAP −1.4153 0.0338 PILRB −1.3019 0.0338 CLEC6A −1.3248 0.0343 PECAM-1 −1.2009 0.0347 PXN −1.329 0.0359 ADGRG1 −1.1823 0.0378 DPP7 −1.1582 0.038 TDRKH −1.2785 0.0385 Siglec-9 −1.1514 0.0387 CD40-L −1.5868 0.0388 VEGFC −1.1727 0.04 LYVE1 −1.227 0.0403 FADD −1.546 0.041 FCRL1 −1.3733 0.0416 EGF −1.7729 0.0419 HGF −1.5542 0.0426 GZMH −1.494 0.0428 CLEC4G −1.1865 0.045 LY75 −1.1401 0.0452 PRDX3 −1.199 0.0465 COL4A1 −1.2699 0.0466 CEACAM8 −1.6177 0.0471 SEMA7A −1.1335 0.0475 NUDT5 −1.5449 0.0476 FCRL6 −1.3556 0.0476 PAPPA −1.3491 0.0485 FASLG −1.3614 0.0486 GRN −1.2448 0.0486 MATN3 −1.3384 0.049

Example 7: Protein Expression Levels for Selected Biomarkers in Complete Responder and Progressive Disease/Death Populations

Targeted proteomic analysis of MCP-3 (CCL7), Reg3A, TNFRSF6B, SCF, CXCL10, IL-8, ST2, CALCA, TNF-R1, IL-6, CCL19, IL-2Ra, and PON3 was conducted using the OLINK proximity extension assay platform. Table 14 provides expression information for each of the proteins within the Complete Responder (CR) and Progressive Disease/Death (PD/Death) groups. For each protein, Table 14 includes the median and mean protein expression levels (pg/ml) within each group, standard error, range, and statistical differences between the CR and PD/Death groups. Statistical differences between the groups were identified using an unpaired T test.

TABLE 14 Protein Expression in Complete Responder and Progressive Disease/Death Populations CR (N = 10) PD/Death (N = 7) p value (pg/ml) (pg/ml) (unpaired Analyte median mean SEM range median mean SEM range t test) MCP-3 2.343 2.613 0.4318 0.7949-4.862  16.66 21.35 5.705 3.907-42.27 0.0013 (CCL7) Reg3A 4150 9252 3597  728.8-28209 49259 47951 8907 19613-81454 0.0006 TNFRSF6B 159 184.7 26.38 67.97-308.7 413.6 411.9 72.41   203-681.7 0.0043 SCF 627.6 639 90.92 185.2-1171  318.4 283.7 38.16 151.2-398.6 0.0071 CXCL10 200.6 307.2 81.94  96.17-879.4 941.8 920.4 231.6 147.6-1991  0.0121 IL-8 5.332 9.346 2.458 2.629-23   42.87 54.49 17.53 15.03-155.7 0.0079 ST2 47037 70902 19650  24630-205075 142056 163519 38569  55016-318173 0.0339 CALCA 1456 1826 392.6 858.5-5026  3130 5996 2494  1375-19759 0.0669 TNF-R1 9199 8822 1056  3713-15630 12659 13129 1265 10015-20208 0.0195 IL-6 1.068 0.9521 0.1724 0.1551-1.791  3.753 7.156 4.24 0.9551-32.2  0.0969 CCL19 439.1 484.9 80.79 156.5-973.4 1036 1751 663.3 203.6-5450  0.0377 IL-2Ra 355 469.8 94.9 63.64-1178  612.6 577.4 96.43 213.8-856.9 0.4771 PON3 386584 438566 61426 254507-885782 93702 148321 37162  50041-284920 0.0025

Other Embodiments

While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. 

What is claimed is:
 1. A method of treating a human subject having Graft-Versus-Host Disease (GvHD), comprising administering to the human subject a therapy comprising a JAK inhibitor, wherein the human subject has been previously determined to have (i) a baseline concentration of at least one protein selected from the group consisting of IL8, HAOX1, ENPP7, ACE2, SULT2A1, MCP-3, CES1, MFGE8, PLXNB1, TNFRSF10A, CCL15, SEMA4C, PREB, NFATC3, CCL19, DLL1, ENTPD2, IL-4RA, EPHA2, FOSB, CXCL10, VAMP5, ALDH3A1, MVK, IL12RB1, CALCA, AHCY, PRSS2, LILRB4, DDAH1, IL-1ra, NECTIN2, PDCD1, CD74, PD-L1, REG3A, CASA, N2DL-2, CDCP1, U-PAR, SIGLEC7, ANGPTL4, ALDH1A1, SPINK1, HTRA2, PRDX6, IL-1RT2, IGFBP-1, HNMT, TRAIL-R2, CXADR, CTSL1, IFN-gamma-R1, IL-18R1, KRT19, KYNU, and TGM2 in a biological sample obtained from the human subject that is lower than a control, and/or (ii) a baseline concentration of at least one protein selected from the group consisting of PON3, CNTN1, IGFBP3, LEP, Notch 3, TN-R, HSD11B1, FAM19A5, NCAN, F11, GDF-8, CCL28, GALNT10, BCAN, TIMP4, CRISP2, CD207, WNT9A, MBL2, EN-RAGE, TWEAK, CR2, MFAP5, KIT, GH, PFKM, CDSN, CRH, GCP5, KLK6, and DRAXIN in a biological sample obtained from the human subject that is higher than a control, wherein the control is the concentration of the protein in a sample or samples obtained from one or more subjects that have not responded to treatment with the JAK inhibitor.
 2. The method of claim 1, wherein the human subject has been previously determined to have (i) a baseline concentration of at least one protein selected from the group consisting of MCP-3, HAOX1, CASA, CALCA, IL8, SULT2A1, VAMP5, SPINK1, ENPP7, ACE2, CTSL1, PRSS2, CXCL10, MFGE8, KRT19, ALDH1A1, CES1, REG3A, KYNU, IL-4RA, CDCP1, MVK, FOSB, NFATC3, N2DL-2, DDAH1, IGFBP-1, ALDH3A1, CXADR, PLXNB1, CD74, ENTPD2, PREB, CCL19, HNMT, HTRA2, IL-1RT2, and IL-18R1 in a biological sample obtained from the human subject that is lower than a control, and/or (ii) a baseline concentration of at least one protein selected from the group consisting of PON3, LEP, MBL2, GH, GDF-8, EN-RAGE, CRISP2, and CR2 in a biological sample obtained from the human subject that is higher than a control.
 3. The method of claim 1, wherein the human subject has been previously determined to have (i) a baseline concentration of at least one protein selected from the group consisting of MCP-3, HAOX1, CASA, CALCA, IL8, SULT2A1, VAMP5, SPINK1, ENPP7, ACE2, CTSL1, PRSS2, CXCL10, MFGE8, KRT19, and ALDH1A1 in a biological sample obtained from the human subject that is lower than a control, and/or (ii) a baseline concentration of at least one protein selected from the group consisting of PON3, LEP, and MBL2 in a biological sample obtained from the human subject that is higher than a control.
 4. The method of claim 1, wherein the human subject has been previously determined to have (i) a baseline concentration of at least one protein selected from the group consisting of MCP-3, HAOX1, CASA, CALCA, IL8, SULT2A1, VAMP5, and SPINK1 in a biological sample obtained from the human subject that is lower than a control, and/or (ii) a baseline concentration of at least one protein selected from the group consisting of PON3 and LEP in a biological sample obtained from the human subject that is higher than a control.
 5. A method of treating a human subject having developing Graft-Versus-Host Disease (GvHD), comprising: providing a biological sample obtained from the human subject; measuring in the biological sample a reduced concentration, as compared to a control, of at least one protein selected from the group consisting of IL8, HAOX1, ENPP7, ACE2, SULT2A1, MCP-3, CES1, MFGE8, PLXNB1, TNFRSF10A, CCL15, SEMA4C, PREB, NFATC3, CCL19, DLL1, ENTPD2, IL-4RA, EPHA2, FOSB, CXCL10, VAMP5, ALDH3A1, MVK, IL12RB1, CALCA, AHCY, PRSS2, LILRB4, DDAH1, IL-1ra, NECTIN2, PDCD1, CD74, PD-L1, REG3A, CASA, N2DL-2, CDCP1, U-PAR, SIGLEC7, ANGPTL4, ALDH1A1, SPINK1, HTRA2, PRDX6, IL-1RT2, IGFBP-1, HNMT, TRAIL-R2, CXADR, CTSL1, IFN-gamma-R1, IL-18R1, KRT19, KYNU, and TGM2, and/or an increased concentration, as compared to a control, of at least one protein selected from the group consisting of PON3, CNTN1, IGFBP3, LEP, Notch 3, TN-R, HSD11B1, FAM19A5, NCAN, F11, GDF-8, CCL28, GALNT10, BCAN, TIMP4, CRISP2, CD207, WNT9A, MBL2, EN-RAGE, TWEAK, CR2, MFAP5, KIT, GH, PFKM, CDSN, CRH, GCP5, KLK6, and DRAXIN; and administering a therapy comprising a JAK inhibitor to the human subject, wherein the control is the concentration of the protein in a sample or samples obtained from one or more subjects that have not responded to treatment with the JAK inhibitor.
 6. The method of claim 5, comprising: measuring in the biological sample a reduced concentration, as compared to a control, of at least one protein selected from the group consisting of MCP-3, HAOX1, CASA, CALCA, IL8, SULT2A1, VAMP5, SPINK1, ENPP7, ACE2, CTSL1, PRSS2, CXCL10, MFGE8, KRT19, ALDH1A1, CES1, REG3A, KYNU, IL-4RA, CDCP1, MVK, FOSB, NFATC3, N2DL-2, DDAH1, IGFBP-1, ALDH3A1, CXADR, PLXNB1, CD74, ENTPD2, PREB, CCL19, HNMT, HTRA2, IL-1RT2, and IL-18R1, and/or an increased concentration, as compared to a control, of at least one protein selected from the group consisting of PON3, LEP, MBL2, GH, GDF-8, EN-RAGE, CRISP2, and CR2; and administering the therapy comprising the JAK inhibitor to the human subject.
 7. The method of claim 5, comprising: measuring in the biological sample a reduced concentration, as compared to a control, of at least one protein selected from the group consisting of MCP-3, HAOX1, CASA, CALCA, IL8, SULT2A1, VAMP5, SPINK1, ENPP7, ACE2, CTSL1, PRSS2, CXCL10, MFGE8, KRT19, and ALDH1A1, and/or an increased concentration, as compared to a control, of at least one protein selected from the group consisting of PON3, LEP, and MBL2; and administering the therapy comprising the JAK inhibitor to the human subject.
 8. The method of claim 5, comprising: measuring in the biological sample a reduced concentration, as compared to a control, of at least one protein selected from the group consisting of MCP-3, HAOX1, CASA, CALCA, IL8, SULT2A1, VAMP5, and SPINK1, and/or an increased concentration, as compared to a control, of at least one protein selected from the group consisting of PON3 and LEP; and administering the therapy comprising the JAK inhibitor to the human subject.
 9. The method of claim 1, wherein a second therapeutic agent is administered to the human subject in combination with the JAK inhibitor.
 10. The method of claim 9, wherein the second therapeutic agent is a corticosteroid, methotrexate, cyclosporine, mycophenolate mofetil, tacrolimus, sirolimus, everolimus, antithymocyte globulin, alemtuzumab, cyclophosphamide, ibrutinib, imatinib, infliximab, etanercept, tocilizumab, alemtuzumab, basiliximab, daclizumab, rituximab, denileukin diftitox, pentostatin, ciclosporin, thalidomide, halofuginone, hydroxychloroquine, or mesenchymal stem cells.
 11. The method of claim 9, wherein the second therapeutic agent is a corticosteroid.
 12. The method of claim 11, wherein the corticosteroid is methylprednisolone or prednisone.
 13. The method of claim 1, wherein the biological sample is blood, serum, plasma, urine, spinal fluid, saliva, lacrimal fluid, or sweat.
 14. The method of claim 1, wherein the biological sample is blood, serum, or plasma.
 15. The method of claim 1, wherein the concentration of the protein is measured by an immunological method.
 16. The method of claim 15, wherein the immunological method is selected from the group consisting of enzyme-linked immunosorbent assay, enzyme immunoassay, radioimmunoassay, chemiluminescent immunoassay, electrochemiluminescence immunoassay, latex turbidimetric immunoassay, latex photometric immunoassay, immuno-chromatographic assay, and western blotting.
 17. The method of claim 1, wherein the concentration of the protein is measured by mass spectrometry.
 18. The method of claim 1, wherein the JAK inhibitor is itacitinib.
 19. The method of claim 1, wherein the JAK inhibitor is 4-[3-(cyanomethyl)-3-(3′,5′-dimethyl-1H, 1′H-4,4′-bipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide or a pharmaceutically acceptable salt thereof or ((2R,5S)-5-{2-[(1R)-1-hydroxyethyl]-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl}tetrahydro-2H-pyran-2-yl)acetonitrile or a pharmaceutically acceptable salt thereof.
 20. The method of claim 1, wherein the GvHD is acute GvHD.
 21. The method of claim 1, wherein the GvHD is chronic GvHD.
 22. The method of claim 5, wherein a second therapeutic agent is administered to the human subject in combination with the JAK inhibitor.
 23. The method of claim 22, wherein the second therapeutic agent is a corticosteroid, methotrexate, cyclosporine, mycophenolate mofetil, tacrolimus, sirolimus, everolimus, antithymocyte globulin, alemtuzumab, cyclophosphamide, ibrutinib, imatinib, infliximab, etanercept, tocilizumab, alemtuzumab, basiliximab, daclizumab, rituximab, denileukin diftitox, pentostatin, thalidomide, halofuginone, hydroxychloroquine, or mesenchymal stem cells.
 24. The method of claim 22, wherein the second therapeutic agent is a corticosteroid.
 25. The method of claim 24, wherein the corticosteroid is methylprednisolone or prednisone.
 26. The method of claim 5, wherein the biological sample is blood, serum, plasma, urine, spinal fluid, saliva, lacrimal fluid, or sweat.
 27. The method of claim 5, wherein the biological sample is blood, serum, or plasma.
 28. The method of claim 5, wherein the concentration of the protein is measured by an immunological method.
 29. The method of claim 28, wherein the immunological method is selected from the group consisting of enzyme-linked immunosorbent assay, enzyme immunoassay, radioimmunoassay, chemiluminescent immunoassay, electrochemiluminescence immunoassay, latex turbidimetric immunoassay, latex photometric immunoassay, immuno-chromatographic assay, and western blotting.
 30. The method of claim 5, wherein the concentration of the protein is measured by mass spectrometry.
 31. The method of claim 5, wherein the JAK inhibitor is itacitinib.
 32. The method of claim 5, wherein the JAK inhibitor is 4-[3-(cyanomethyl)-3-(3′,5′-dimethyl-1H, 1′H-4,4′-bipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro-N-[(1 S)-2,2,2-trifluoro-1-methylethyl]benzamide or a pharmaceutically acceptable salt thereof or ((2R,5S)-5-{2-[(1R)-1-hydroxyethyl]-1H-imidazo[4,5-d]thieno [3,2-b]pyridin-1-yl}tetrahydro-2H-pyran-2-yl)acetonitrile or a pharmaceutically acceptable salt thereof.
 33. The method of claim 5, wherein the GvHD is acute GvHD.
 34. The method of claim 5, wherein the GvHD is chronic GvHD. 